Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks.

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Sorkio, Anni; Koch, Lothar; Koivusalo, Laura; Deiwick, Andrea; Miettinen, Susanna; Chichkov, Boris; Skottman, Heli

2018-07-01

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted

Progress in corneal wound healing

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Ljubimov, Alexander V.; Saghizadeh, Mehrnoosh

2015-01-01

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and

Effects of artificial tear treatment on corneal epithelial thickness and corneal topography findings in dry eye patients.

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Çakır, B; Doğan, E; Çelik, E; Babashli, T; Uçak, T; Alagöz, G

2018-05-01

To investigate the effects of artificial tear treatment on central corneal epithelial thickness, and central, mid-peripheral and peripheral corneal thicknesses in patients with dry eye disease (DED). Patients with DED underwent ocular examinations, including Schirmer-2 test, slit lamp examination for tear break-up time (BUT), corneal topography (CT) for measuring mean central, mid-peripheral and peripheral corneal thickness values and anterior segment optic coherence tomography (AS-OCT) for obtaining central corneal epithelial thickness. After artificial tear treatment (carboxymethylcellulose and sodium hyaluronate formulations) for one month, patients were examined again at a second visit and the results were compared. Sixty-one eyes of 33 female dry eye patients (mean age: 38.3±5.7 years) were enrolled. The mean follow-up time was 36.4±3.3 days. The mean tear BUT and Schirmer-1 tests revealed significant improvement after treatment (P=0.000, P=0.000, respectively). Central corneal epithelium and mean mid-peripheral corneal thicknesses measured significantly higher after treatment (P=0.001, P=0.02). Changes in central and peripheral corneal thicknesses were not statistically significant. Artificial tear treatment in dry eye patients seems to increase central corneal epithelial and mid-peripheral corneal thicknesses. Measurement of corneal epithelial thickness can be a useful tool for evaluation of treatment response in dry eye patients. Further long-term prospective studies are needed to investigate this item. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns

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Cejka, Cestmir; Holan, Vladimir; Trosan, Peter; Zajicova, Alena; Javorkova, Eliska; Cejkova, Jitka

2016-01-01

The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties. PMID:27057279

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns.

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Cejka, Cestmir; Holan, Vladimir; Trosan, Peter; Zajicova, Alena; Javorkova, Eliska; Cejkova, Jitka

2016-01-01

The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns

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Cestmir Cejka

2016-01-01

Full Text Available The aim of this study was to examine whether mesenchymal stem cells (MSCs and/or corneal limbal epithelial stem cells (LSCs influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs, with adipose tissue MSCs (Ad-MSCs, or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9, inducible nitric oxide synthase (iNOS, α-smooth muscle actin (α-SMA, transforming growth factor-β1 (TGF-β1, and vascular endothelial factor (VEGF were low. The central corneal thickness (taken as an index of corneal hydration increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.

Corneal Epithelial Remodeling and Its Effect on Corneal Asphericity after Transepithelial Photorefractive Keratectomy for Myopia

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Jie Hou

2016-01-01

Full Text Available Purpose. To evaluate the changes in epithelial thickness profile following transepithelial photorefractive keratectomy (T-PRK for myopia and to investigate the effect of epithelial remodeling on corneal asphericity. Methods. Forty-four patients (44 right eyes who underwent T-PRK were retrospectively evaluated. Epithelial thickness was measured using spectral-domain optical coherence tomography at different corneal zones (central, 2 mm; paracentral, 2–5 mm; and mid-peripheral, 5-6 mm preoperatively and at 1 week and 1, 3, and 6 months postoperatively. The correlation between the changes in corneal epithelial thickness (ΔCET and postoperative Q-value changes (ΔQ was analyzed 6 months postoperatively. Results. Epithelial thickness at 6 months showed a negative meniscus-like lenticular pattern with less central thickening, which increased progressively toward the mid-periphery (3.69±4.2, 5.19±3.8, and 6.23±3.9 μm at the center, paracenter, and mid-periphery, resp., P<0.01. A significant positive relationship was observed between epithelial thickening and ΔQ 6 months postoperatively (r=0.438, 0.580, and 0.504, resp., P<0.01. Conclusions. Significant epithelial thickening was observed after T-PRK and showed a lenticular change with more thickening mid-peripherally, resulting in increased oblateness postoperatively. Epithelial remodeling may modify the epithelial thickness profile after surface ablation refractive surgery for myopia.

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation.

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Shi, Long; Chen, Hongmei; Yu, Xiaoming; Wu, Xinyi

2013-11-01

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.

Do topical antibiotics help corneal epithelial trauma?

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King, J. W.; Brison, R. J.

1993-01-01

Topical antibiotics are routinely used in emergency rooms to treat corneal trauma, although no published evidence supports this treatment. In a noncomparative clinical trial, 351 patients with corneal epithelial injuries were treated without antibiotics. The infection rate was 0.7%, suggesting that such injuries can be safely and effectively managed without antibiotics. A comparative clinical trial is neither warranted nor feasible.

CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

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After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

Healing of corneal epithelial wounds in marine and freshwater fish.

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Ubels, J L; Edelhauser, H F

The corneal epithelium of a fish is in direct contact with the aquatic environment and is a barrier to movement of ions and water into and through the cornea. This tissue layer is thus important in maintenance of corneal transparency. When the epithelium is wounded, its protective function is lost and corneal transparency remains compromised until the epithelial barrier is re-established. This study was undertaken to investigate the healing response of the fish cornea to epithelial abrasion. Wounds were stained with fluorescein and photographed during healing. Wound areas were measured by planimetry. The cornea of the sculpin, a marine teleost, becomes edematous after wounding and heals at 2.54 to 3.42 mm2/hr. Nonswelling corneas of the elasmobranchs--dogfish shark and skate--heal at 1.29 mm2/hr, respectively. The wounded eye of the rainbow trout, a freshwater teleost, is stressed by the low osmolality of the environment. Severe corneal edema and cataracts develop following epithelial wounding, and the cornea heals at 0.64 mm2/hr. Although the healing rates in teleosts differ from those in mammals, histology shows that the corneal healing mechanism is essentially the same in fish and mammals.

Corneal surface reconstruction - a short review

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Madhavan H N

2009-01-01

Full Text Available Cornea is the clear, dome-shaped surface that covers the front of the eye and when damage due to burns or injury and several other diseases, stem cells residing in its rim called "limbus" are stimulated to multiply to support growth of new epithelial cells over its surface. If this ready source of stem cells is damaged or destroyed the natural repair is not possible and such a condition is known as corneal limbal stem cell deficiency (CLSCD disease. Stem cell transplant helps such persons to regenerate the corneal surface. Human corneal limbal stem cell transplantation is at present an established procedure with reasonable good clinical outcome particularly when autologous limbal epithelial tissue from a fellow unaffected eye is used. 1, 2 A major concern related to the autograft is the possibility of CLSCD at the donor site, 3 techniques that allowed the expansion of a small limbal biopsy in the laboratory using cell cultures that could be then transplanted to the affected eye have been developed ,4, 5 Human amniotic membrane (HAM is used as a scaffold for both culturing the human limbal epithelial cells and for ocular surface reconstruction with the cultured limbal epithelial cells. 4-7 However, researchers have used alternative scaffolds like collagen 8, fibrin gel 9 and cross-linked gel of fibronectin and fibrin. 10 All these are biological materials and also need for animal 3T3 feeder layer for stem cell cultures. The properties of HAM are unique including antiadhesive effects, bacteriostatic effects, wound protection, pain reduction, and improvement of epithelialization and characteristically lacking imunogenicity. The use of amniotic membrane transplantation (AMT to treat ocular surface abnormalities was first reported by Graziella Pellegrini, chief of stem cell laboratory at Giovanni Paolo Hospital in Venice, Italy, who was the first to demonstrate the limbal stem cell transplant in 1997. Amniotic membrane has been successfully used in

Pathophysiology of Corneal Scarring in Persistent Epithelial Defects After PRK and Other Corneal Injuries.

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Wilson, Steven E; Medeiros, Carla S; Santhiago, Marcony R

2018-01-01

To analyze corneal persistent epithelial defects that occurred at 3 to 4 weeks after -4.50 diopter (D) photorefractive keratectomy (PRK) in rabbits and apply this pathophysiology to the treatment of persistent epithelial defects that occur after any corneal manipulations or diseases. Two of 168 corneas that had -4.50 D PRK to study epithelial basement membrane regeneration developed spontaneous persistent epithelial defects that did not heal at 3 weeks after PRK. These were studied with slit-lamp photographs, immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin (α-SMA), and transmission electron microscopy. Myofibroblasts developed at the stromal surface within the persistent epithelial defect and for a short distance peripheral to the leading edge of the epithelium. No normal epithelial basement membrane was detectable within the persistent epithelial defect or for up to 0.3 mm behind the leading edge of the epithelium, although epithelial basement membrane had normally regenerated in other areas of the zone ablated by an excimer laser where the epithelium healed promptly. A persistent epithelial defect in the cornea results in the development of myofibroblasts and disordered extracellular matrix produced by these cells that together cause opacity within, and a short distance beyond, the persistent epithelial defect. Clinicians should treat persistent epithelial defects within 10 days of non-closure of the epithelium to facilitate epithelial healing to prevent long-term stromal scarring (fibrosis). [J Refract Surg. 2018;34(1):59-64.]. Copyright 2018, SLACK Incorporated.

Assessment of corneal epithelial thickness in dry eye patients.

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Cui, Xinhan; Hong, Jiaxu; Wang, Fei; Deng, Sophie X; Yang, Yujing; Zhu, Xiaoyu; Wu, Dan; Zhao, Yujin; Xu, Jianjiang

2014-12-01

To investigate the features of corneal epithelial thickness topography with Fourier-domain optical coherence tomography (OCT) in dry eye patients. In this cross-sectional study, 100 symptomatic dry eye patients and 35 normal subjects were enrolled. All participants answered the ocular surface disease index questionnaire and were subjected to OCT, corneal fluorescein staining, tear breakup time, Schirmer 1 test without anesthetic (S1t), and meibomian morphology. Several epithelium statistics for each eye, including central, superior, inferior, minimum, maximum, minimum - maximum, and map standard deviation, were averaged. Correlations of epithelial thickness with the symptoms of dry eye were calculated. The mean (±SD) central, superior, and inferior corneal epithelial thickness was 53.57 (±3.31) μm, 52.00 (±3.39) μm, and 53.03 (±3.67) μm in normal eyes and 52.71 (±2.83) μm, 50.58 (±3.44) μm, and 52.53 (±3.36) μm in dry eyes, respectively. The superior corneal epithelium was thinner in dry eye patients compared with normal subjects (p = 0.037), whereas central and inferior epithelium were not statistically different. In the dry eye group, patients with higher severity grades had thinner superior (p = 0.017) and minimum (p dry eye corneal epithelium was thinner than normal eyes in the superior region. In more severe dry eye disease patients, the superior and minimum epithelium was much thinner, with a greater range of map standard deviation.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

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Aberdam, Edith; Petit, Isabelle; Sangari, Linda; Aberdam, Daniel

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC as a cellular alternative for in vitro ocular toxicity testing.

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Edith Aberdam

Full Text Available Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

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Bhavani S. Kowtharapu

2018-02-01

Full Text Available Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM on corneal epithelial cell function along with substance P (SP. Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK, paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained

Efficacy of highly hydrophilic soft contact lenses for persistent corneal epithelial defects after anterior segment surgery

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Zhi-Wei Peng

2015-02-01

Full Text Available AIM:To investigate the efficacy of highly hydrophilic soft contact lenses for persistent corneal epithelial defects.METHODS:In this retrospective case analysis, 28 patients(28 eyeswith persistent corneal epithelial defects after anterior segment surgery from January 2011 to June 2013 in our hospital were reviewed. After regular treatment for at least 2wk, the persistent corneal epithelial defects were treated with highly hydrophilic soft contact lenses, until the corneal epithelial healing. Continued to wear the same lens no more than 3wk, or in need of replacement the new one. All cases were followed up for 6mo. Key indicators of corneal epithelial healling, corneal fluorescein staining and ocular symptoms improvement were observed.RESULTS: Twenty-one eyes were cured(75.00%, markedly effective in 5 eyes(17.86%, effective in 2 eyes(7.14%, no invalid cases, the total efficiency of 100.00%. Ocular symptoms of 25 cases(89.29%relieved within 2d, the rest 3 cases(10.71%relieved within 1wk. The corneal epithelial of 6 cases(21.43%repaired in 3wk, 13 cases(46.43%in 6wk, 7 cases(25.00%in 9wk, 2 cases(7.14%over 12wk. There were no signs of secondary infection. And no evidence of recurrence in 6mo. CONCLUSION: Highly hydrophilic soft contact lenses could repair persistent corneal epithelial defects after anterior segment surgery significantly, while quickly and effectively relieve a variety of ocular irritation.

Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study.

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Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu

2014-01-01

Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds.

Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

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Chi-Chin Sun

Full Text Available Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

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Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

2010-02-19

14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

14-3-3σ controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

International Nuclear Information System (INIS)

Xin, Ying; Lu, Qingxian; Li, Qiutang

2010-01-01

14-3-3σ (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3σ mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3σ activity in corneal epithelial cells by overexpressing dominative negative 14-3-3σ led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3σ mutant-expressing corneal epithelial cells. We conclude that 14-3-3σ is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

Polysaccharide hydrogel combined with mesenchymal stem cells promotes the healing of corneal alkali burn in rats.

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Yifeng Ke

Full Text Available Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β, antiangiogenic cytokine (TSP-1 and decrease those promoting inflammation (TNF-α, chemotaxis (MIP-1α and MCP-1 and angiogenesis (VEGF and MMP-2. This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.

Polysaccharide Hydrogel Combined with Mesenchymal Stem Cells Promotes the Healing of Corneal Alkali Burn in Rats

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Liu, Xun; Yu, Min; Yang, Chunbo; Li, Xiaorong

2015-01-01

Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs) have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs) were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β), antiangiogenic cytokine (TSP-1) and decrease those promoting inflammation (TNF-α), chemotaxis (MIP-1α and MCP-1) and angiogenesis (VEGF and MMP-2). This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder. PMID:25789487

Spontaneous regression of epithelial downgrowth from clear corneal phacoemulsification wound

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Ryan M. Jaber

2018-06-01

Full Text Available Purpose: To report a case of spontaneous regression of optical coherence tomography (OCT and confocal microscopy-supported epithelial downgrowth associated with clear corneal phacoemulsification wound. Observations: A 66-year-old Caucasian male presented two years after phacoemulsification in the left eye with an enlarging cornea endothelial lesion in that eye. His early post-operative course had been complicated by corneal edema and iris transillumination defects. The patient presented to our clinic with a large geographic sheet of epithelial downgrowth and iris synechiae to the temporal clear corneal wound. His vision was correctable to 20/25 in his left eye. Anterior segment OCT showed a hyperreflective layer on the posterior cornea with an abrupt transition that corresponded to the clinical transition zone of the epithelial downgrowth. Confocal microscopy showed polygonal cells with hyperreflective nuclei suggestive of epithelial cells in the area of the lesion with a transition to a normal endothelial cell mosaic. Given the lack of glaucoma or inflammation and the relatively good vision, the plan was made to closely monitor for progression with the anticipation that he may require aggressive surgery. Over course of subsequent follow-up visits at three, seven and ten months; the endothelial lesion receded significantly. Confocal imaging in the area of the previously affected cornea showed essentially normal morphology with anan endothelial cell count of 1664 cells/mm2. Conclusions and importance: Epithelial downgrowth may spontaneously regress. Though the mechanism is yet understood, contact inhibition of movement may play a role. Despite this finding, epithelial downgrowth is typically a devastating process requiring aggressive treatment. Keywords: Epithelial downgrowth, Spontaneous regression, Confocal microscopy, Contact inhibition of movement

Changes in Matrix Metalloproteinases in Diabetes Patients' Tears After Vitrectomy and the Relationship With Corneal Epithelial Disorder.

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Matsumura, Takehiro; Takamura, Yoshihiro; Tomomatsu, Takeshi; Arimura, Shogo; Gozawa, Makoto; Takihara, Yuji; Inatani, Masaru

2015-06-01

Previous studies indicate involvement of matrix metalloproteinases (MMPs) in the pathogenesis of diabetic keratopathy. To evaluate MMP levels in the tears of patients with diabetes, we investigated changes in MMP levels during perioperative periods and clarify the relationship with corneal epithelial disorders following vitrectomy. Matrix metalloproteinase levels in tears were measured by multiplex bead array in patients with or without diabetes who were scheduled for vitrectomy. Twenty-two patients with diabetes and proliferative diabetic retinopathy, and 20 patients with epiretinal membrane or macular hole (control group), were recruited. Changes in MMP levels during perioperative periods and the relationship with corneal epithelial disorders after vitrectomy were analyzed. The levels of MMP-2, -9, and -10 at 1 day after surgery in the diabetic group were significantly higher than in the control group. At 1 week after surgery, MMP-10 levels in the diabetic group were significantly higher than in the control group. After vitrectomy, corneal epithelial disorders occurred in six patients in the diabetic group but not in the control group. In the diabetic group, MMP-10 levels in tears of patients with corneal epithelial disorders were significantly higher than those in patients without corneal epithelial disorders. The MMP concentration in tears of patients with diabetes was higher than in nondiabetic patients after vitrectomy. High MMP-10 levels were observed in patients with diabetes and corneal epithelial disorders after vitrectomy. Aberrant levels of MMP-10 may cause corneal epithelial disorder after vitrectomy.

Cellular toxicity of calf blood extract on human corneal epithelial cells in vitro.

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Park, Young Min; Kim, Su Jin; Han, Young Sang; Lee, Jong Soo

2015-01-01

To investigate the biologic effects of the calf blood extract on corneal epithelial cells in vitro. The effects on corneal epithelial cells were evaluated after 1, 4, 12, and 24 h of exposure to various concentrations of calf blood extract (3, 5, 8 and 16%). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was performed to measure levels of cellular metabolic activity. The lactate dehydrogenase (LDH) assay was performed to determine the extent of cellular damage. Cellular morphology was examined using phase-contrast microscopy. The scratch wound assay was performed to quantify the migration of corneal epithelial cells. At the 3 and 5% concentrations of calf blood extract, MTT values were similar to those observed in the control group. However, at a concentration of 8 and 16%, cellular metabolic activity was significantly decreased after 4 h of exposure to calf blood extract. After 12 h of exposure to 8 and 16% concentrations of calf blood extract, LDH activity and cellular morphological damage to the corneal epithelial cells were significantly increased. There was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract because of cellular toxicity. Compared with normal corneal epithelial cells, the cellular activity was decreased, and toxicity was increased after over 12 h of exposure to more than 5% concentration of calf blood extract. Further clinical studies will be necessary to determine the optimal concentration and exposure time for the topical application of eye drops containing calf blood extract.

Metaherpetic corneal disease in a dog associated with partial limbal stem cell deficiency and neurotrophic keratitis.

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Ledbetter, Eric C; Marfurt, Carl F; Dubielzig, Richard R

2013-07-01

To describe clinical, in vivo confocal microscopic, histopathologic, and immunohistochemical features of a dog with metaherpetic corneal disease that developed subsequent to a protracted episode of canine herpesvirus-1 (CHV-1) dendritic ulcerative keratitis. A 7-year-old, spayed-female, Miniature Schnauzer was treated for bilateral CHV-1 dendritic ulcerative keratitis. Following resolution of ulcerative keratitis, sectoral peripheral superficial corneal gray opacification, vascularization, and pigmentation slowly migrated centripetally to the axial cornea of both eyes. Corneal sensitivity measured with a Cochet-Bonnet esthesiometer was dramatically and persistently reduced. In vivo corneal confocal microscopic examination revealed regions of epithelium with a conjunctival phenotype. In these areas, the surface epithelium was thin, disorganized, and composed of hyper-reflective epithelial cells. Goblet cells and Langerhans cells were frequent, and the subbasal nerve plexus was completely absent or markedly diminished. Histopathologic abnormalities in the globes were restricted to the superficial cornea and included sectoral corneal conjunctivalization, increased anterior stromal spindle cells, and vascularization. Immunohistochemical evaluation of the corneas with anti-neurotublin antibody demonstrated attenuation of the epithelial and subbasal nerve plexuses with marked stromal hyperinnervation and increased numbers of morphologically abnormal neurites. Similar to herpes simplex virus keratitis in humans, CHV-1 ulcerative keratitis may be associated with the development of chronic degenerative corneal disease in dogs. In the described dog, this chronic corneal disease included progressive corneal opacification because of partial limbal stem cell deficiency and neurotrophic keratitis. Long-term monitoring of dogs following resolution of active CHV-1 keratitis may be indicated, particularly when ulcerations persist for an extended period. © 2012 American College of

Breakdown evaluation of corneal epithelial barrier caused by antiallergic eyedrops using an electrophysiologic method.

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Nakashima, Mikiro; Nakamura, Tadahiro; Teshima, Mugen; To, Hideto; Uematsu, Masafumi; Kitaoka, Takashi; Taniyama, Kotaro; Nishida, Koyo; Nakamura, Junzo; Sasaki, Hitoshi

2008-02-01

The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those in other appraisal methods. Six kinds of antiallergic eyedrops, including benzalkonium chloride (BK) as an ophthalmic preservative and two kinds of BK-free antiallergic eyedrops, were used in this study. Eyedrops were applied to excise rabbit corneas and monitoring was performed according to an electrophysiologic method, using a commercially available chamber system to mimic human tear turnover. Changes in transepithelial electrical resistance (TEER) in the corneal surface were recorded. The cytotoxicity of each kind of eyedrops in a normal rabbit corneal epithelial (NRCE) cell line and a human endothelial cell line EA.hy926 was also examined. The extent of decrease in the corneal TEER after applying antiallergic eyedrops was dependent on the concentration of the BK included as a preservative, but it was also affected by the different kinds of drugs when the BK concentration was low. Higher cytotoxicity of the eyedrops against the NRCE and EA.hy926 cell lines was observed with a reduction of TEER. Monitoring changes in the corneal TEER, according to the electrophysiologic method with the application of antiallergic eyedrops, is useful for predicting corneal epithelial breakdown caused by their instillation.

Fluorescent in situ hybridization (FISH) on corneal impression cytology specimens (CICS): study of epithelial cell survival after keratoplasty.

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Catanese, Muriel; Popovici, Cornel; Proust, Hélène; Hoffart, Louis; Matonti, Frédéric; Cochereau, Isabelle; Conrath, John; Gabison, Eric E

2011-02-22

To assess corneal epithelial cell survival after keratoplasty. Corneal impression cytology (CIC) was performed on sex-mismatched corneal transplants. Fluorescent in situ hybridization (FISH) with sex chromosome-specific probes was performed to identify epithelial cell mosaicism and therefore allocate the donor or recipient origin of the cells. Twenty-four samples of corneal epithelial cells derived from 21 transplanted patients were analyzed. All patients received post-operative treatment using dexamethasone eye drops, with progressive tapering over 18 months, and nine patients also received 2% cyclosporine eye drops. Out of the 24 samples reaching quality criteria, sex mosaicism was found in 13, demonstrating the presence of donor-derived cells at the center of the graft for at least 211 days post keratoplasty. Kaplan-Meier analysis established a median survival of donor corneal epithelial cells of 385 days. Although not statistically significant, the disappearance of donor cells seemed to be delayed and the average number of persistent cells appeared to be greater when 2% cyclosporine was used topically as an additional immunosuppressive therapy. The combination of corneal impressions and FISH analysis is a valuable tool with negligible side effects to investigate the presence of epithelial cell mosaicism in sex-mismatched donor transplants. Epithelial cells survived at the center of the graft with a median survival of more than one year, suggesting slower epithelial turnover than previously described.

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

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Chang Rae Rho

Full Text Available Granulocyte-macrophage colony-stimulating factor (GM-CSF is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs. We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF. An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml. MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

Protective effects of trehalose on the corneal epithelial cells.

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Aragona, Pasquale; Colosi, Pietro; Rania, Laura; Colosi, Francesca; Pisani, Antonina; Puzzolo, Domenico; Micali, Antonio

2014-01-01

Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Twelve patients undergoing laser subepithelial keratomileusis (LASEK) were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. In both trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE), the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

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Kim, Tae-Hyun; Park, Young-Woo; Ahn, Jae-Sang; Ahn, Jeong-Taek; Kim, Se-Eun; Jeong, Man-Bok; Seo, Min-Su; Kang, Kyung-Sun

2013-01-01

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits. PMID:23388445

Role of Bone Morphogenetic Protein 7 (BMP7 in the Modulation of Corneal Stromal and Epithelial Cell Functions

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Bhavani S. Kowtharapu

2018-05-01

Full Text Available In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we

Highly concentrated collagen solutions leading to transparent scaffolds of controlled three-dimensional organizations for corneal epithelial cell colonization.

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Tidu, Aurélien; Ghoubay-Benallaoua, Djida; Teulon, Claire; Asnacios, Sophie; Grieve, Kate; Portier, François; Schanne-Klein, Marie-Claire; Borderie, Vincent; Mosser, Gervaise

2018-05-29

This study aimed at controlling both the organization and the transparency of dense collagen scaffolds making use of the lyotropic mesogen properties of collagen. Cholesteric or plywood-like liquid crystal phases were achieved using mixtures of acetic and hydrochloric acids as solvents. The critical pH at which the switch between the two phases occurred was around pH = 3. The use of the two acids led to fibrillated collagen I scaffolds, whose visual aspect ranged from opaque to transparent. Rheological investigations showed that viscoelastic properties of the plywood-like solutions were optimized for molding due to faster recovery. They also confirmed the correlation between the elastic modulus and the diameter of collagen fibrils obtained after fibrillogenesis under ammonia vapor. Human corneal epithelial cells, grown from donor limbal explants, were cultured both on transparent plywood-like matrices and on human amniotic membranes for 14 days. The development of corneal epithelium and the preservation of epithelial stem cells were checked by optical microscopy, colony formation assay, immuno-fluorescence and quantitative polymerase chain reaction. A higher level of amplification of limbal stem cells was obtained with collagen matrices compared with amniotic membranes, showing the high biocompatibility of our scaffolds. We therefore suggest that collagen solutions presenting both plywood-like organization and transparency might be of interest for biomedical applications in ophthalmology.

The SULFs, extracellular sulfatases for heparan sulfate, promote the migration of corneal epithelial cells during wound repair.

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Inna Maltseva

Full Text Available Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS. SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1⁻/⁻, but not Sulf2⁻/⁻, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1⁻/⁻ mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE. Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

Protective Effects of Trehalose on the Corneal Epithelial Cells

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Pasquale Aragona

2014-01-01

Full Text Available Purpose. Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Methods. Twelve patients undergoing laser subepithelial keratomileusis (LASEK were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. Results. In both trehalose-untreated eyes (TUE and trehalose-treated eyes (TTE, the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Conclusions. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

Nodular Epithelial Hyperplasia after Photorefractive Keratectomy Followed by Corneal Collagen Cross-Linking

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Bogoni, Ayla; Salerno, Liberdade Cezaro; Ghanem, Vinícius Coral; Ghanem, Ramon Coral

2013-01-01

This study describes a case of nodular epithelial hyperplasia and stromal alterations in a patient with keratoconus who was submitted to topography-guided photorefractive keratectomy (PRK) followed by corneal collagen cross-linking. Debridement of the epithelial nodule was performed. After a 2-year followup, a new topography-guided PRK was indicated.

Treatment with solubilized Silk-Derived Protein (SDP enhances rabbit corneal epithelial wound healing.

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Waleed Abdel-Naby

Full Text Available There is a significant clinical need to improve current therapeutic approaches to treat ocular surface injuries and disease, which affect hundreds of millions of people annually worldwide. The work presented here demonstrates that the presence of Silk-Derived Protein (SDP on the healing rabbit corneal surface, administered in an eye drop formulation, corresponds with an enhanced epithelial wound healing profile. Rabbit corneas were denuded of their epithelial surface, and then treated for 72-hours with either PBS or PBS containing 5 or 20 mg/mL SDP in solution four times per day. Post-injury treatment with SDP formulations was found to accelerate the acute healing phase of the injured rabbit corneal epithelium. In addition, the use of SDP corresponded with an enhanced tissue healing profile through the formation of a multi-layered epithelial surface with increased tight junction formation. Additional biological effects were also revealed that included increased epithelial proliferation, and increased focal adhesion formation with a corresponding reduction in the presence of MMP-9 enzyme. These in vivo findings demonstrate for the first time that the presence of SDP on the injured ocular surface may aid to improve various steps of rabbit corneal wound healing, and provides evidence that SDP may have applicability as an ingredient in therapeutic ophthalmic formulations.

Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells: the protective effects of lactoferrin

International Nuclear Information System (INIS)

Shimmura, Shigeto; Suematsu, Makoto; Shimoyama, Masaru; Oguchi, Yoshihisa; Ishimura, Yuzuru

1996-01-01

Acute exposure to suprathreshold ultraviolet B radiation (UV-B) is known to cause photokeratitis resulting from the necrosis and shedding of corneal epithelial cells. However, the corneal effects of low dose UV-B in the environmental range is less clear. In this study, subthreshold UV-B was demonstrated to cause non-necrotic peroxide formation in cultured corneal epithelial cells, which was attenuated by the major tear protein lactoferrin. Intracellular oxidative insults and cell viability of rabbit corneal epithelial cells (RCEC) were assessed by dual-color digital microfluorography using carboxydichlorofluorescin (CDCFH) diacetate bis (acetoxymethyl) ester, a hydroperoxide-sensitive fluoroprobe, and propidium iodode (PI) respectively. The magnitude of UV-induced oxidative insults was calibrated by concentrations of exogenously applied H 2 O 2 which evoke compatible levels of CDCFH oxidation. Exposure of RCEC to low-dose UV-B (2.0 mJ cm -2 at 313 nm, 10.0 mJ cm -2 total UV-B) caused intracellular oxidative changes which were equivalent to those elicited by 240 μM hydrogen peroxide under the conditions of the study. The changes were dose dependent, non-necrotic, and were partially inhibited by lactoferrin ( 1 mg ml -1 ) but not by iron-saturated lactoferrin. Pretreatment with deferoxamine (2 mΜ) or catalase (100 U ml -1 ) also attenuated the UV-induced oxidative stress. The results indicate that UV-B comparable to solar irradiation levels causes significant intracellular peroxide formation in corneal epithelial cells, and that lactoferrin in tears may have a physiological role in protecting the corneal epithelium from solar UV irradiation. (Author)

Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

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Barbaro, Vanessa; Nasti, Annamaria Assunta; Raffa, Paolo; Migliorati, Angelo; Nespeca, Patrizia; Ferrari, Stefano; Palumbo, Elisa; Bertolin, Marina; Breda, Claudia; Miceli, Francesco; Russo, Antonella; Caenazzo, Luciana; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

2016-08-01

: Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative

Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.

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Hongshan Liu

Full Text Available BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Limbal Stem Cell Deficiency and Treatment with Stem Cell Transplantation.

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Barut Selver, Özlem; Yağcı, Ayşe; Eğrilmez, Sait; Gürdal, Mehmet; Palamar, Melis; Çavuşoğlu, Türker; Ateş, Utku; Veral, Ali; Güven, Çağrı; Wolosin, Jose Mario

2017-10-01

The cornea is the outermost tissue of the eye and it must be transparent for the maintenance of good visual function. The superficial epithelium of the cornea, which is renewed continuously by corneal stem cells, plays a critical role in the permanence of this transparency. These stem cells are localized at the cornea-conjunctival transition zone, referred to as the limbus. When this zone is affected/destroyed, limbal stem cell deficiency ensues. Loss of limbal stem cell function allows colonization of the corneal surface by conjunctival epithelium. Over 6 million people worldwide are affected by corneal blindness, and limbal stem cell deficiency is one of the main causes. Fortunately, it is becoming possible to recover vision by autologous transplantation of limbal cells obtained from the contralateral eye in unilateral cases. Due to the potential risks to the donor eye, only a small amount of tissue can be obtained, in which only 1-2% of the limbal epithelial cells are actually limbal stem cells. Vigorous attempts are being made to expand limbal stem cells in culture to preserve or even enrich the stem cell population. Ex vivo expanded limbal stem cell treatment in limbal stem cell deficiency was first reported in 1997. In the 20 years since, various protocols have been developed for the cultivation of limbal epithelial cells. It is still not clear which method promotes effective stem cell viability and this remains a subject of ongoing research. The most preferred technique for limbal cell culture is the explant culture model. In this approach, a small donor eye limbal biopsy is placed as an explant onto a biocompatible substrate (preferably human amniotic membrane) for expansion. The outgrowth (cultivated limbal epithelial cells) is then surgically transferred to the recipient eye. Due to changing regulations concerning cell-based therapy, the implementation of cultivated limbal epithelial transplantation in accordance with Good Laboratory Practice using

A novel collagen film with micro-rough surface structure for corneal epithelial repair fabricated by freeze drying technique

International Nuclear Information System (INIS)

Liu, Yang; Ren, Li; Wang, Yingjun

2014-01-01

Highlights: • Collagen film with micro-rough surface is fabricated by freeze drying technique. • The film has suitable water uptake capability and toughness performance. • The film has good optical performance. • Human corneal epithelial cells studies confirmed the biocompatibility of the film. - Abstract: Corneal epithelial defect is a common disease and keratoplasty is a common treatment method. A collagen film with micro-rough surface was fabricated through a simple freeze drying technique in this study. Compared with the air-dried collagen film (AD-Col), this freeze-dried collagen film (FD-Col) has a more suitable water uptake capability (about 85.5%) and toughness performance. Both of the two films have good optical properties and the luminousness of them is higher than 80%. Besides, the adhesion and proliferation rate of human corneal epithelial cells on the micro-rough surface of FD-Col film is higher than that on the smooth surface of AD-Col film. The results indicate that this FD-Col film may have potential applications for corneal epithelial repair

A novel collagen film with micro-rough surface structure for corneal epithelial repair fabricated by freeze drying technique

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Liu, Yang [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wang, Yingjun, E-mail: imwangyj@163.com [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China)

2014-05-01

Highlights: • Collagen film with micro-rough surface is fabricated by freeze drying technique. • The film has suitable water uptake capability and toughness performance. • The film has good optical performance. • Human corneal epithelial cells studies confirmed the biocompatibility of the film. - Abstract: Corneal epithelial defect is a common disease and keratoplasty is a common treatment method. A collagen film with micro-rough surface was fabricated through a simple freeze drying technique in this study. Compared with the air-dried collagen film (AD-Col), this freeze-dried collagen film (FD-Col) has a more suitable water uptake capability (about 85.5%) and toughness performance. Both of the two films have good optical properties and the luminousness of them is higher than 80%. Besides, the adhesion and proliferation rate of human corneal epithelial cells on the micro-rough surface of FD-Col film is higher than that on the smooth surface of AD-Col film. The results indicate that this FD-Col film may have potential applications for corneal epithelial repair.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

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Jing Dan

2017-03-01

Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

Experiment of amnion epithelial cell suspension liquid used for acute rabbit corneal alkali burn

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Yan-Yan Zhang

2017-10-01

Full Text Available AIM: To investigate the effects of amnion epithelial cell(AECsuspension liquid on the biological behavior of the rabbit's corneal epithelium, combined with the in vitro and in vivo experiments. METHODS: The rabbit's corneal epithelium were cultured in the lower chamber of transwell, and AEC suspension liquid was dropwised in the upper chamber. There was only culture medium in the upper chamber of the control group. The proliferation of rabbit's corneal epithelium was observed with CCK-8 automated colorimetry and the expression of PCNA was detected by immunocytochemistry. We used the scratch wound assay to detect the migration of corneal epithelial cell(CEC. The in vivo models were established by placing a 10mm diameter corneal trephine in the center of the cornea, within 1mol/L NaOH for 1min. We divided those into three groups: treatment group of AEC suspension liquid eye drop, AEC suspension liquid subconjunctival injection and the control group without any treatment. Using the slit-lamp biomicroscope and fluorescence staining to observe the cornea per week. After 28d we took the eyeballs with the HE staining. The expression of VEGF was detected by immunohistochemistry. RESULTS: The activity of CEC with AEC treatment was much higher than the control group(PPIn vivo, the inflammation of the corneal and the CNV of the AEC group were all significantly reduced compared with the control group(PPCONCLUSION: AEC suspension liquid can promote the proliferation and migration of the rabbit's corneal epithelium. The potential of AEC suspension liquid as a therapy for acute corneal alkali burn.

Keratolimbal autograft transplantation as a possible new treatment of Lisch epithelial corneal dystrophy.

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Celis Sánchez, J; Mesa Varona, D V; Avendaño Cantos, E; López-Romero Moraleda, S; Cebrian Rosado, E; González Del Valle, F

2016-07-01

The case concerns 64-year-old woman with visual acuity of 20/40 in the right eye. Slit-lamp examination revealed a grey, feathery corneal opacification with intraepithelial microcysts compatible with Lisch epithelial corneal dystrophy (LECD). It was treated with epithelial debridements, contact lenses and mitomycin C, but the opacification recurred within months. The removal of limbus sector and autologous limbal transplantation (KLAT) were used successfully without recurrence. After removal of damaged limbus, KLAT should be considered as a treatment option for asymmetric LECD when other treatments have failed. Copyright © 2016 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.

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Espana, Edgar M; Di Pascuale, Mario A; He, Hua; Kawakita, Tetsuya; Raju, Vadrevu K; Liu, Chia-Yang; Tseng, Scheffer C G

2004-09-01

To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker. Copyright Association for Research in Vision and Ophthalmology

In vitro effects of three blood derivatives on human corneal epithelial cells.

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Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

2012-08-15

We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

The Use of an IL-1 Receptor Antagonist Peptide to Control Inflammation in the Treatment of Corneal Limbal Epithelial Stem Cell Deficiency

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E. Fok

2015-01-01

Full Text Available Corneal limbal stem cell deficiency (LSCD may be treated using ex vivo limbal epithelial stem cells (LESCs derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation, and transplant rejection. Lipopolysaccharide (LPS or recombinant human IL-1β stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation. The peptide was characterized using mass spectroscopy and high performance liquid chromatography. Peptide cytotoxicity, patterns of cell cytokine expression in response to LPS or IL-1β stimulation, and peptide suppression of this response were investigated by MTS/LDH assays, ELISA, and q-PCR. Cell differences in LPS stimulated toll-like receptor 4 expression were investigated using immunocytochemistry. A significant reduction in rIL-1β stimulated inflammatory cytokine production occurred following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1 mg/mL P<0.05. LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.

A minimal model of epithelial tissue dynamics and its application to the corneal epithelium

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Henkes, Silke; Matoz-Fernandez, Daniel; Kostanjevec, Kaja; Coburn, Luke; Sknepnek, Rastko; Collinson, J. Martin; Martens, Kirsten

Epithelial cell sheets are characterized by a complex interplay of active drivers, including cell motility, cell division and extrusion. Here we construct a particle-based minimal model tissue with only division/death dynamics and show that it always corresponds to a liquid state with a single dynamic time scale set by the division rate, and that no glassy phase is possible. Building on this, we construct an in-silico model of the mammalian corneal epithelium as such a tissue confined to a hemisphere bordered by the limbal stem cell zone. With added cell motility dynamics we are able to explain the steady-state spiral migration on the cornea, including the central vortex defect, and quantitatively compare it to eyes obtained from mice that are X-inactivation mosaic for LacZ.

Myogel supports the ex-vivo amplification of corneal epithelial cells.

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Francis, D; Abberton, K; Thompson, E; Daniell, M

2009-03-01

Limbal stem cell deficiency leads to conjunctivalisation of the cornea and subsequent loss of vision. The recent development of transplantation of ex-vivo amplified corneal epithelium, derived from limbal stem cells, has shown promise in treating this challenging condition. The purpose of this research was to compare a variety of cell sheet carriers for their suitability in creating a confluent corneal epithelium from amplified limbal stem cells. Cadaveric donor limbal cells were cultured using an explant technique, free of 3T3 feeder cells, on a variety of cell sheet carriers, including denuded amniotic membrane, Matrigel, Myogel and stromal extract. Comparisons in rate of growth and degree of differentiation were made, using immunocytochemistry (CK3, CK19 and ABCG2). The most rapid growth was observed on Myogel and denuded amniotic membrane, these two cell carriers also provided the most reliable substrata for achieving confluence. The putative limbal stem cell marker, ABCG2, stained positively on cells grown over Myogel and Matrigel but not for those propagated on denuded amniotic membrane. In the clinical setting amniotic membrane has been demonstrated to provide a suitable carrier for limbal stem cells and the resultant epithelium has been shown to be successful in treating limbal stem cell deficiency. Myogel may provide an alternative cell carrier with a further reduction in risk as it is has the potential to be derived from an autologous muscle biopsy in the clinical setting.

Intraoperative corneal thickness measurements during corneal collagen cross-linking with isotonic riboflavin solution without dextran in corneal ectasia.

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Cınar, Yasin; Cingü, Abdullah Kürşat; Sahin, Alparslan; Türkcü, Fatih Mehmet; Yüksel, Harun; Caca, Ihsan

2014-03-01

Abstract Objective: To monitor the changes in corneal thickness during the corneal collagen cross-linking procedure by using isotonic riboflavin solution without dextran in ectatic corneal diseases. The corneal thickness measurements were obtained before epithelial removal, after epithelial removal, following the instillation of isotonic riboflavin solution without dextran for 30 min, and after 10 min of ultraviolet A irradiation. Eleven eyes of eleven patients with progressive keratoconus (n = 10) and iatrogenic corneal ectasia (n = 1) were included in this study. The mean thinnest pachymetric measurements were 391.82 ± 30.34 µm (320-434 µm) after de-epithelialization of the cornea, 435 ± 21.17 µm (402-472 µm) following 30 min instillation of isotonic riboflavin solution without dextran and 431.73 ± 20.64 µm (387-461 µm) following 10 min of ultraviolet A irradiation to the cornea. Performing corneal cross-linking procedure with isotonic riboflavin solution without dextran might not induce corneal thinning but a little swelling throughout the procedure.

Effect of basic fibroblast growth factor and cytochrome c peroxidase combination in transgenic mice corneal epithelial healing process after excimer laser photoablation

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Sergio Zaccaria Scalinci

2011-02-01

Full Text Available Sergio Zaccaria Scalinci1, Lucia Scorolli1, Alessandro Meduri2, Pier Luigi Grenga3, Giulia Corradetti1, Cristian Metrangolo11Low Vision Center – University of Bologna, Bologna, Italy; 2Department of Surgical Specialities, Ophthalmology Clinic, University of Messina, Messina, Italy; 3Department of Ophthalmology, University of Rome "La Sapienza", Rome, ItalyPurpose: To evaluate the role of prepared basic fibroblast growth factor (bFGF and cytochrome c peroxidase (CCP combination eyedrops in corneal epithelial healing of transgenic mice (B6(A-Rperd12/J after excimer laser photoablation. Materials and methods: In this prospective study, 216 eyes of 108 mice underwent bilateral photorefractive keratectomy. We considered 4 groups: A, B, C, and D. Group A received standard topical postoperative therapy with tobramycin, diclofenac, and dexamethasone eyedrops plus CCP at 3 drops per day for a week or until corneal re-epithelialization was achieved. Group B received standard topical postoperative therapy plus bFGF eyedrops and phosphate-buffered saline (PBS 3 drops per day for a week or until corneal re-epithelialization was complete. In group C, 1 eye received standard topical postoperative therapy plus CCP eyedrops, bFGF eyedrops, and PBS 3 drops per day for a week or until corneal re-epithelialization was complete. Control eyes (group D received a standard topical postoperative therapy plus placebo eyedrops. Mice were followed-up for a week from the day after the surgery to evaluate the rate of corneal re-epithelialization.Results: Data were analyzed by ANOVA using the XLSTAT 2010 software. Eyes in group A, B, and C healed completely before the fifth postoperative day, achieving, respectively, a re-epithelialization time of 92 hours ± 10 SD, 90 hours ± 12 SD, and 86 hours ± 12 SD. Group D had a re-epithelialization time of 121 hours ± 8 SD (P < 0.05. No side effects or toxic effects were documented.Conclusions: Results suggest that re-epithelialization

Changes in corneal sensation, epithelial damage, and tear function after descemet stripping automated endothelial keratoplasty.

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Hirayama, Yumiko; Satake, Yoshiyuki; Hirayama, Masatoshi; Shimazaki-Den, Seika; Konomi, Kenji; Shimazaki, Jun

2013-09-01

To study the ocular surface changes in eyes after Descemet stripping automated endothelial keratoplasty (DSAEK) compared with those after penetrating keratoplasty (PKP). This prospective study compared the changes in 31 eyes of 28 patients who underwent DSAEK (DSAEK group) with those in 15 disease-matched eyes of 15 patients who underwent PKP (PKP group). Corneal epithelial integrity was evaluated using a fluorescein staining score. Corneal sensation was measured with a Cochet-Bonnet esthesiometer. Tear function was evaluated using the Schirmer test, tear clearance test, tear function index, and tear break-up time. The postoperative fluorescein staining score was significantly higher in the PKP group than in the DSAEK group (P = 0.02). Postoperative corneal sensation was significantly better in the DSAEK group than in the PKP group (P sensation after DSAEK was significantly better than the preoperative value (P = 0.02). There were no statistically significant changes in the Schirmer test, tear clearance test, tear function index, or break-up time before and after the surgery in both the DSAEK and PKP groups. No significant differences were observed between the DSAEK and PKP groups after the surgery. Corneal sensation was preserved, and epithelial damage was less severe after DSAEK compared with PKP. Preservation of corneal sensation may contribute to the early recovery of visual function and long-term maintenance of ocular surface health after DSAEK.

Corneal regeneration by induced human buccal mucosa cultivated on an amniotic membrane following alkaline injury.

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Man, Rohaina Che; Yong, Then Kong; Hwei, Ng Min; Halim, Wan Haslina Wan Abdul; Zahidin, Aida Zairani Mohd; Ramli, Roszalina; Saim, Aminuddin Bin; Idrus, Ruszymah Binti Hj

2017-01-01

Various clinical disorders and injuries, such as chemical, thermal, or mechanical injuries, may lead to corneal loss that results in blindness. PURPOSE : The aims of this study were to differentiate human buccal mucosa (BMuc) into corneal epithelial-like cells, to fabricate engineered corneal tissue using buccal mucosal epithelial cells, and to reconstruct a damaged corneal epithelium in a nude rat model. BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages. Corneal stem cell markers β1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct. In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

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Petznick, Andrea; Madigan, Michele C; Garrett, Qian; Sweeney, Deborah F; Evans, Margaret D M

2013-01-01

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (pTears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following

Regeneration of defective epithelial basement membrane and restoration of corneal transparency

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Marino, Gustavo K.; Santhiago, Marcony R.; Santhanam, Abirami; Torricelli, Andre A. M.; Wilson, Steven E.

2018-01-01

PURPOSE To study regeneration of the normal ultrastructure of the epithelial basement membrane (EBM) in rabbit corneas that had -9D photorefractive keratectomy (PRK) and developed late haze (fibrosis) with restoration of transparency over one to four months after surgery and in corneas that had incisional wounds. METHODS Twenty-four rabbits had one of their eyes included into one of the two procedure groups (-9D PRK or nearly full-thickness incisional wounds), while the opposite eye serving as unwounded controls. All corneas were evaluated with slit lamp photos, transmission electron microscopy and immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin and collagen type III. RESULTS In the ‘-9D PRK group’, corneas at one month after surgery had dense corneal haze and no evidence of regenerated EBM ultrastructure. By two months after surgery, however, small areas of stromal clearing began to appear within the confluent opacity (lacunae), and these corresponded to small islands of normally-regenerated EBM detected within larger area of the excimer laser-ablated zone with no evidence of normal EBM. By four months after surgery, the EBM was fully-regenerated and the corneal transparency was completely restored to the ablated zone. In the ‘Incisional wound group’, the two dense, linear corneal opacities were observed at one month after surgery and progressively faded by two and three months after surgery. The EBM ultrastructure was fully regenerated at the site of the incisions, including around epithelial plugs that extended into the stroma, by one month after surgery in all eyes. CONCLUSIONS In the rabbit model, spontaneous resolution of corneal fibrosis (haze) after high correction PRK is triggered by regeneration of EBM with normal ultrastructure in the excimer laser- ablated zone. Conversely, incisional wounds heal in rabbit corneas without the development of myofibroblasts because the EBM regenerates normally by one month after surgery

Assessment of Corneal Epithelial Thickness in Asymmetric Keratoconic Eyes and Normal Eyes Using Fourier Domain Optical Coherence Tomography

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S. Catalan

2016-01-01

Full Text Available Purpose. To compare the characteristics of asymmetric keratoconic eyes and normal eyes by Fourier domain optical coherence tomography (OCT corneal mapping. Methods. Retrospective corneal and epithelial thickness OCT data for 74 patients were compared in three groups of eyes: keratoconic (n=22 and normal fellow eyes (n=22 in patients with asymmetric keratoconus and normal eyes (n=104 in healthy subjects. Areas under the curve (AUC of receiver operator characteristic (ROC curves for each variable were compared across groups to indicate their discrimination capacity. Results. Three variables were found to differ significantly between fellow eyes and normal eyes (all p<0.05: minimum corneal thickness, thinnest corneal point, and central corneal thickness. These variables combined showed a high discrimination power to differentiate fellow eyes from normal eyes indicated by an AUC of 0.840 (95% CI: 0.762–0.918. Conclusions. Our findings indicate that topographically normal fellow eyes in patients with very asymmetric keratoconus differ from the eyes of healthy individuals in terms of their corneal epithelial and pachymetry maps. This type of information could be useful for an early diagnosis of keratoconus in topographically normal eyes.

Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

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Li, W; Chen, Y-T; Hayashida, Y; Blanco, G; Kheirkah, A; He, H; Chen, S-Y; Liu, C-Y; Tseng, SCG

2010-01-01

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens–Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP–Pax6 transgenes into these Pax6− clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium. PMID:18027901

Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition.

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Rush, Jamie S; Bingaman, David P; Chaney, Paul G; Wax, Martin B; Ceresa, Brian P

2016-11-01

The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 μM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition. In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology. Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation. An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.

Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells.

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Yu-Chieh Wu

Full Text Available BACKGROUND: Type I insulin-like growth factor receptor (IGF-1R and insulin receptor (INSR are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R. The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium. METHODOLOGY/PRINCIPLE FINDINGS: IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways. CONCLUSION/SIGNIFICANCE: In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic

Effect of topical vitamin E on ethanol-induced corneal epithelial apoptosis.

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Bilgihan, Kamil; Konuk, Onur; Hondur, Ahmet; Akyürek, Nalan; Ozogul, Candan; Hasanreisoglu, Berati

2005-01-01

Ethanol is used to loosen the corneal epithelium before photoablation in laser subepithelial keratomileusis (LASEK). In this study, the apoptotic index of corneal epithelium after ethanol exposure and the effects of topical vitamin E were evaluated. The study was performed on 28 rabbit eyes in four groups. Group 1 comprised the controls. In group 2, 20% ethanol was applied topically for 20 seconds. In group 3, topical vitamin E was applied following 20% ethanol application. In group 4, only topical vitamin E was applied. Apoptosis was evaluated with TUNEL assay and transmission electron microscopy. Epithelial apoptosis was detected in all specimens in group 2. No apoptosis was detected in other groups except for one eye in group 1. The apoptotic index in group 2 was statistically higher than other groups (P < .001).

Corneal Densitometry as a Tool to Measure Epithelial Ingrowth After Laser In Situ Keratomileusis.

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Adran, Daniel; Vaillancourt, Louis; Harissi-Dagher, Mona; Kruh, Jonathan N; Syed, Zeba A; Robinson, Steven; Melki, Samir

2017-04-01

This study evaluates the correlation between corneal densitometry and epithelial ingrowth (EI) after laser in situ keratomileusis (LASIK). Corneal densitometry of 3 patients who developed EI after LASIK was measured with the Oculus Pentacam. Corneal densitometry readings of each patient were obtained preoperatively and postoperatively after ingrowth was discovered. Densitometry was recorded at the central nest of opacity and at the leading edges of EI. For all patients, the most severe stages of EI observed on slit-lamp photographs correlated with the highest densitometry readings, with peak densitometry ranging from 73.3 to 95.1. These values were much higher than preoperative densitometry readings, which ranged from 21.8 to 27.2. In 2 cases, the Pentacam densitometry map revealed progression of EI toward the visual axis that was only faintly detectable or not detectable at all on the corresponding slit-lamp photographs. Corneal densitometry seems to be an objective measure of the severity and progression of EI after LASIK.

Evaluation of Corneal Neovascularization Using Optical Coherence Tomography Angiography in Patients With Limbal Stem Cell Deficiency.

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Oie, Yoshinori; Nishida, Kohji

2017-11-01

Detection of the exact area of corneal neovascularization using slit-lamp photography is often difficult. Thus, we evaluated corneal neovascularization in patients with limbal stem cell deficiency using optical coherence tomography angiography (OCTA). Five patients with 5 eyes showing partial or total limbal stem cell deficiency were enrolled. Three eyes had severe corneal scarring. Five 6- × 6-mm images (frontal, upper, lower, nasal, and temporal) were obtained by OCTA. Slit-lamp photography was performed for all patients on the same day. OCTA has 2 advantages over slit-lamp photography for clear demonstration of corneal neovascularization. First, OCTA can show neovascularization in cases with severe corneal opacification. Second, OCTA can detect not only large vessels but also small vessels that cannot be seen by slit-lamp photography. OCTA is a powerful tool for objective evaluation of vascularization in the anterior and posterior segments of the eye. We have demonstrated that OCTA can visualize corneal neovascularization in patients with corneal diseases more clearly than slit-lamp photography.

Recovery of the sub-basal nerve plexus and superficial nerve terminals after corneal epithelial injury in mice.

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Downie, Laura E; Naranjo Golborne, Cecilia; Chen, Merry; Ho, Ngoc; Hoac, Cam; Liyanapathirana, Dasun; Luo, Carol; Wu, Ruo Bing; Chinnery, Holly R

2018-06-01

Our aim was to compare regeneration of the sub-basal nerve plexus (SBNP) and superficial nerve terminals (SNT) following corneal epithelial injury. We also sought to compare agreement when quantifying nerve parameters using different image analysis techniques. Anesthetized, female C57BL/6 mice received central 1-mm corneal epithelial abrasions. Four-weeks post-injury, eyes were enucleated and processed for PGP9.5 to visualize the corneal nerves using wholemount immunofluorescence staining and confocal microscopy. The percentage area of the SBNP and SNT were quantified using: ImageJ automated thresholds, ImageJ manual thresholds and manual tracings in NeuronJ. Nerve sum length was quantified using NeuronJ and Imaris. Agreement between methods was considered with Bland-Altman analyses. Four-weeks post-injury, the sum length of nerve fibers in the SBNP, but not the SNT, was reduced compared with naïve eyes. In the periphery, but not central cornea, of both naïve and injured eyes, nerve fiber lengths in the SBNP and SNT were strongly correlated. For quantifying SBNP nerve axon area, all image analysis methods were highly correlated. In the SNT, there was poor correlation between manual methods and auto-thresholding, with a trend towards underestimating nerve fiber area using auto-thresholding when higher proportions of nerve fibers were present. In conclusion, four weeks after superficial corneal injury, there is differential recovery of epithelial nerve axons; SBNP sum length is reduced, however the sum length of SNTs is similar to naïve eyes. Care should be taken when selecting image analysis methods to compare nerve parameters in different depths of the corneal epithelium due to differences in background autofluorescence. Copyright © 2018 Elsevier Ltd. All rights reserved.

Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2) and glutamatergic receptors.

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Oswald, Duane J; Lee, Albert; Trinidad, Monique; Chi, Cheryl; Ren, Ruiyi; Rich, Celeste B; Trinkaus-Randall, Vickery

2012-01-01

Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+) wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+) mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+) mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+) waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA) receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2 and glutamatergic receptors.

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Duane J Oswald

Full Text Available Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2 receptors resulting in mobilization of a Ca(2+ wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+ wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+ mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+ mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+ waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

Resolvin D1 promotes corneal epithelial wound healing and restoration of mechanical sensation in diabetic mice.

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Zhang, Zhenzhen; Hu, Xiaoli; Qi, Xia; Di, Guohu; Zhang, Yangyang; Wang, Qian; Zhou, Qingjun

2018-01-01

To investigate the effect and mechanism of proresolving lipid mediator resolvin D1 (RvD1) on the corneal epithelium and the restoration of mechanical sensation in diabetic mice. Type 1 diabetes was induced in mice with intraperitoneal streptozocin injections. The healthy and diabetic mice underwent removal of the central corneal epithelium, and then 100 ng/ml RvD1 or its formyl peptide receptor 2 (FPR2) antagonist WRW4 was used to treat the diabetic mice. Regeneration of the corneal epithelium and nerves was observed with sodium fluorescein staining and whole-mount anti-β3-tubulin fluorescence staining. The inflammatory response level was measured with hematoxylin and eosin staining (inflammatory cell infiltration), enzyme-linked immunosorbent assay (tumor necrosis factor alpha and interleukin-1 beta content), myeloperoxidase activity, and fluorescence staining (macrophage content). The reactive oxygen species (ROS) and glutathione (GSH) levels were examined with incubation with fluorescent probes, and oxidative stress-related protein expression levels were evaluated with fluorescence staining and western blotting. Topical application of RvD1 promoted regeneration of the corneal epithelium in diabetic mice, accompanied by the reactivation of signaling and inflammation resolution related to regeneration of the epithelium. Furthermore, RvD1 directly attenuated the accumulation of ROS and nicotinamide adenine dinucleotide phosphate oxidase 2/4 expression, while RvD1 enhanced GSH synthesis and reactivated the Nrf2-ARE signaling pathway that was impaired in the corneal epithelium in the diabetic mice. More interestingly, topical application of RvD1 promoted regeneration of corneal nerves and completely restored impaired mechanical sensitivity of the cornea in diabetic mice. In addition, the promotion of corneal epithelial wound healing by RvD1 in diabetic mice was abolished by its FPR2 antagonist WRW4. Topical application of RvD1 promotes corneal epithelial wound

Generation of a TALEN-mediated, p63 knock-in in human induced pluripotent stem cells.

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Kobayashi, Yuki; Hayashi, Ryuhei; Quantock, Andrew J; Nishida, Kohji

2017-12-01

The expression of p63 in surface ectodermal cells during development of the cornea, skin, oral mucosa and olfactory placodes is integral to the process of cellular self-renewal and the maintenance of the epithelial stem cell status. Here, we used TALEN technology to generate a p63 knock-in (KI) human induced pluripotent stem (hiPS) cell line in which p63 expression can be visualized via enhanced green fluorescent protein (EGFP) expression. The KI-hiPS cells maintained pluripotency and expressed the stem cell marker gene, ΔNp63α. They were also able to successfully differentiate into functional corneal epithelial cells as assessed by p63 expression in reconstructed corneal epithelium. This approach enables the tracing of p63-expressing cell lineages throughout epithelial development, and represents a promising application in the field of stem cell research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Gefarnate stimulates mucin-like glycoprotein secretion in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models

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Dota A

2013-01-01

Full Text Available Atsuyoshi Dota, Yuko Takaoka-Shichijo, Masatsugu NakamuraOphthalmic Research and Development Center, Santen Pharmaceutical Co, Ltd, Ikoma-shi, Nara, JapanPurpose: The aim of this study was to evaluate the effect of gefarnate on mucin-like glycoprotein secretion in isolated rabbit conjunctival tissue, and on corneal epithelial damage in rabbit and cat dry-eye models.Methods: Conjunctival tissue isolated from rabbits was treated with gefarnate. Mucin-like glycoprotein was detected in the culture supernatant by an enzyme-linked lectin assay. Gefarnate ointment was topically applied to eyes once daily for 7 days in the rabbit dry-eye model, in which the lacrimal glands, Harderian gland, and nictitating membrane were removed, or for 4 weeks in the cat dry-eye model, in which the lacrimal gland and nictitating membrane were removed. Corneal epithelial damage was evaluated by measurement of corneal permeability by rose bengal in the rabbit model or by fluorescein staining in the cat model.Results: Gefarnate stimulated mucin-like glycoprotein secretion in conjunctival tissue in a dose-dependent manner. In the rabbit dry-eye model, application of gefarnate ointment to the eyes resulted in a dose-dependent decrease in rose bengal permeability in the cornea, with the effect being significant at concentrations of ≥0.3%. In the cat dry-eye model, application of gefarnate ointment resulted in a significant decrease in the corneal fluorescein staining score.Conclusion: These results suggest that gefarnate stimulates in vitro secretion of mucin-like glycoprotein in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models. Gefarnate may therefore be effective for treating dry eye.Keywords: gefarnate, fluorescein staining, rose bengal permeability, rabbit, cat, dry eye

[Cell-based therapies - an innovative therapeutic option in ophthalmology: Treating corneal diseases with stem cells].

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Bakker, Ann-Christin; Langer, Barbara

2015-11-01

Pathological changes and disorders of the cornea are a major cause of severe visual impairment and blindness. Replacement of a pathologically altered cornea with healthy corneal tissue from the eye of a suitable donor is among the most common and successful transplantation procedures in medicine. In Germany, approximately 5000-6000 corneal transplantations are performed each year, but the total demand per year is estimated to be twice as high. With a success rate of 90%, the outcome of cornea transplantation is very favourable. However, long-term maintenance and regeneration of a healthy new cornea requires tissue-specific corneal stem cells residing at the basal layer of the limbus, which is the annular transition zone between the cornea and sclera. When this important limbal stem cell population is destroyed or dysfunctional, a pathological condition known as limbal stem cell deficiency (LSCD) manifests. Limbal stem cell deficiency describes conditions associated with impaired corneal wound healing and regeneration. In this situation, transplantation of healthy limbal stem cells is the only curative treatment approach for restoration of an intact and functional ocular surface. To date, treatment of LSCD presents a great challenge for ophthalmologists. However, innovative, cell-therapeutic approaches may open new, promising treatment perspectives. In February 2015, the European Commission granted marketing authorization to the first stem cell-based treatment in the European Union. The product named Holoclar® is an advanced therapy medicinal product (ATMP) for the treatment of moderate to severe LSCD due to physical and chemical burns in adults. Further cell-based treatment approaches are in clinical development.

EXPERIMENTAL REPAIR OF DEEP CORNEAL DEFECTS USING A BIO-CONSTRUCT COMPRISING A COLLAGEN TYPE I MATRIX LOADED WITH BUCCAL EPITHELIAL CELLS

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N. S. Egorova

2017-01-01

Full Text Available The  research  objective was  to study the  reparative effects of  the  collagen  type  I bio-construct loaded  with buccal epithelial cells, on the rabbit cornea after experimental keratectomy at various stages of treatment (on the 3rd, 7th, 14th, 3 0th days.Material  and methods.  The  experiments were  conducted on 20 rabbits  of  the  Chinchilla breed that  were  operated on cornea of both eyes aiming to inflict epithelial and stromal cornea defects. The collagen-based bio-construct bearing buccal epithelial cells was placed  over the cornea of the experimental eyes. The  cornea of the control  eyes was covered with smooth contact lens. After the surgery, a temporal blepharorrhaphy was performed and kept for 3 days. We studied macroand microscopic pattern of corneal regeneration at 3, 7, 14, and 30 days of experiment.Results. When  using the collaged-based bio-construct containing buccal epithelial cells, the complete epithelialization of the corneal defect occurred at mean 7 days earlier compared to that in the control eyes. Thus, the offered bio-construct stimulated the cell migration and proliferation at early stages of treatment (3–7 days reducing the inflammation activity.Conclusion. The bio-construct comprising a collagen type  I matrix loaded with buccal epithelial cells can provide an effective treatment option for corneal defects.

Lingual Epithelial Stem Cells and Organoid Culture of Them

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Hiroko Hisha

2016-01-01

Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing

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Natural killer cells are lymphocytes of the innate immune system that have crucial cytotoxic and regulatory roles in adaptive immunity and inflammation. Herein, we consider a role for these cells in corneal wound healing. After a 2-mm central epithelial abrasion of the mouse cornea, a subset of clas...

The Protective Role of Hyaluronic Acid in Cr(VI-Induced Oxidative Damage in Corneal Epithelial Cells

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Wei Wu

2017-01-01

Full Text Available Cr(VI exposure could produce kinds of intermediates and reactive oxygen species, both of which were related to DNA damage. Hyaluronan (HA has impressive biological functions and was reported to protect corneal epithelial cells against oxidative damage induced by ultraviolet B, benzalkonium chloride, and sodium lauryl sulfate. So the aim of our study was to investigate HA protection on human corneal epithelial (HCE cells against Cr(VI-induced toxic effects. The HCE cell lines were exposed to different concentrations of K2Cr2O7 (1.875, 3.75, 7.5, 15.0, and 30 μM or a combination of K2Cr2O7 and 0.2% HA and incubated with different times (15 min, 30 min, and 60 min. Our data showed that Cr(VI exposure could cause decreased cell viability, increased DNA damage, and ROS generation to the HCE cell lines. But incubation of HA increased HCE cell survival rates and decreased DNA damage and ROS generation induced by Cr(VI in a dose- and time-dependent manner. We report for the first time that HA can protect HCE cells against the toxicity of Cr(VI, indicating that it will be a promising therapeutic agent to corneal injuries caused by Cr(VI.

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

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Ayaki, Masahiko; Yaguchi, Shigeo; Iwasawa, Atsuo; Koide, Ryohei

2008-08-01

The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

ICAM-1 is necessary for epithelial recruitment of gammadelta T cells and efficient corneal wound healing.

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Wound healing and inflammation are both significantly reduced in mice that lack gammadelta T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in gammadelta T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epi...

Ocular surface changes in limbal stem cell deficiency caused by chemical injury: a histologic study of excised pannus from recipients of cultured corneal epithelium.

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Fatima, A; Iftekhar, G; Sangwan, V S; Vemuganti, G K

2008-09-01

To report histopathologic changes of the ocular surface pannus in patients with severe limbal stem cell deficiency (LSCD). Corneal and conjunctival pannus tissues from 29 patients undergoing ocular reconstruction with cultured limbal cell transplantation were included. The medical records of these patients were reviewed for demographics, aetiologic diagnosis, type of injury, interval between the initial insult and excision of pannus, and medical history involving human amniotic membrane (HAM) or limbal transplantation. The paraffin-embedded tissues were reviewed for epithelial changes, type-degree of fibrosis, degenerative changes, vascular changes, conjunctivalization of corneal surface, and evidence of residual HAM. We attempted a clinicopathologic correlation to understand the pathogenesis of pannus formation in LSCD. The 29 tissues were from 29 eyes of patients with primary aetiology of chemical burn in 89.6% (undetermined in 10.4%) of cases. The pannus showed epithelial hyperplasia in 62%, active fibrosis in 66%, severe inflammation in 21%, giant cell reaction in 28%, and stromal calcification in 14% cases. Goblet cells were seen over the cornea in 64% cases; their absence was associated with squamous metaplasia of the conjunctiva and with long duration of insult. Evidence of residual HAM was noted in 42% cases. The commonest cause of severe LSCD is alkali-induced injury. Goblet cells over the cornea were seen in 60% of cases. HAM used for ocular surface reconstruction could persist for long periods within the corneal pannus, thus raising the need for further studies with long-term follow-up.

Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.

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Sawazaki, Ryoichi; Ishihara, Tomoaki; Usui, Shinya; Hayashi, Erika; Tahara, Kayoko; Hoshino, Tatsuya; Higuchi, Akihiro; Nakamura, Shigeru; Tsubota, Kazuo; Mizushima, Tohru

2014-04-21

Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

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Djida Ghoubay-Benallaoua

Full Text Available Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC and epithelial limbal (LSC stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8, E8 supplemented with EGF (E8+ or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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Kathryn L McCabe

Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

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Shuxian Jiang

2010-03-01

Full Text Available Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid

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Zhao, Xuan; Liu, Yang; Li, Weichang; Long, Kai; Wang, Lin; Liu, Sa; Wang, Yingjun; Ren, Li

2015-01-01

Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)–citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col–CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS = 60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30 ± 5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin–eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col–CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications. - Highlights: • Adding different amounts of citric acid could change the properties of films. • The crosslinked films had better mechanical property than non-modified films. • Crosslinked collagen–citric acid films could tolerate suture during operation. • The films showed good ability of epithelial and stromal repair

Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid

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Zhao, Xuan; Liu, Yang; Li, Weichang; Long, Kai; Wang, Lin; Liu, Sa; Wang, Yingjun [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China)

2015-10-01

Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)–citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col–CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS = 60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30 ± 5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin–eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col–CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications. - Highlights: • Adding different amounts of citric acid could change the properties of films. • The crosslinked films had better mechanical property than non-modified films. • Crosslinked collagen–citric acid films could tolerate suture during operation. • The films showed good ability of epithelial and stromal repair.

Corneal epithelial alterations resulting from use of chlorine-disinfected contact tonometer after myopic photorefractive keratectomy.

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Maldonado, M J

1998-08-01

This study aimed to describe a previously unreported complication associated with the use of chlorine-disinfected applanation tonometer heads for intraocular pressure measurement after excimer laser photorefractive keratectomy. Two retrospective case reports. Two patients underwent, respectively, a 7-diopter and a 4-diopter myopic excimer laser correction in their first eye 2 weeks apart. Complete epithelial closure of the ablated area was observed by biomicroscopy in the first-week examination. Four weeks after photorefractive keratectomy, a complete ophthalmic examination was performed. Goldmann applanation tonometry was performed bilaterally after thoroughly rinsing and drying the tonometer biprism, which had been immersed regularly in a chlorine 5000-parts per million solution. Slit-lamp examination and corneal topographic surface regularity were measured. A few minutes after applanation tonometry, both patients reported ocular discomfort in the excimer laser-treated eyes, whereas the untreated fellow eyes were painless. Punctate corneal lesions and superficial epithelial cell clumping were present in the first patient's treated eye, predominantly in the inferior aspect of the applanated cornea. Visual inspection showed a normal tonometer tip. In the second patient's treated cornea, a focal epithelial defect was identified biomicroscopically, which corresponded to the steeper region within the ablation zone on the videokeratograph. In this case, crystal deposits were found on the tonometer tip. The epithelial alterations resolved without sequelae in both cases. Disinfecting solutions of chlorine can cause crystal deposit formation on the tonometer head. Applanation tonometry after repeated disinfection with chlorine solutions appears to have the potential for disrupting the epithelial layer of the healing cornea. Covered contact tonometry or noncontact tonometry should be evaluated as alternative methods to chemically disinfected contact tonometry for

Pre-Clinical Cell-Based Therapy for Limbal Stem Cell Deficiency.

Science.gov (United States)

Sehic, Amer; Utheim, Øygunn Aass; Ommundsen, Kristoffer; Utheim, Tor Paaske

2015-08-28

The cornea is essential for normal vision by maintaining transparency for light transmission. Limbal stem cells, which reside in the corneal periphery, contribute to the homeostasis of the corneal epithelium. Any damage or disease affecting the function of these cells may result in limbal stem cell deficiency (LSCD). The condition may result in both severe pain and blindness. Transplantation of ex vivo cultured cells onto the cornea is most often an effective therapeutic strategy for LSCD. The use of ex vivo cultured limbal epithelial cells (LEC), oral mucosal epithelial cells, and conjunctival epithelial cells to treat LSCD has been explored in humans. The present review focuses on the current state of knowledge of the many other cell-based therapies of LSCD that have so far exclusively been explored in animal models as there is currently no consensus on the best cell type for treating LSCD. Major findings of all these studies with special emphasis on substrates for culture and transplantation are systematically presented and discussed. Among the many potential cell types that still have not been used clinically, we conclude that two easily accessible autologous sources, epidermal stem cells and hair follicle-derived stem cells, are particularly strong candidates for future clinical trials.

Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs

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Keping Xu

2011-02-01

Full Text Available Keping Xu1, Mark McDermott1, Linda Villanueva2, Rhett M Schiffman2, David A Hollander21The Kresge Eye Institute, Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI, USA; 2Allergan, Inc., Irvine, CA, USAPurpose: Ketorolac 0.45% is a new formulation of topical ketorolac in which preservative (benzalkonium chloride, BAK was removed and carboxymethylcellulose (CMC was added to improve tolerability and reduce dosing frequency. This study compared the effects of ketorolac 0.45% on corneal wound healing to prior ketorolac formulations (0.4% and 0.5%, bromfenac 0.09%, and nepafenac 0.1%.Methods: Two parallel-group comparisons were performed in series. A 5-mm central epithelial wound was made in fresh porcine corneas. After 24 hours in minimum essential medium (MEM, corneas were incubated for 10 minutes with study drugs, Triton X-100 1% (positive control, or MEM (negative control, followed by 24 hours in MEM. The remaining wound area was stained, photographed, and quantified (pixels. Study 1 compared ketorolac 0.45% to ketorolac 0.4% and ketorolac 0.5%. Study 2 compared ketorolac 0.45% to bromfenac 0.09% and nepafenac 0.1%.Results: The mean (±SD original wound area was 200,506 ± 4,363 pixels, which was reduced to 59,509 ± 4850 at 48 hours after exposure to Triton X-100 1%. In study 1, the mean remaining wound areas at 48 hours in pixels were 2969 ± 1633 with MEM, 586 ± 299 with ketorolac 0.45% (significantly reduced, P < 0.05 vs all other treatments, 10,228 ± 7541 with ketorolac 0.4%, and 50,674 ± 33,409 with ketorolac 0.5% (significantly enlarged, P < 0.05 vs MEM. In study 2, the mean remaining wound areas at 48 hours were 565 ± 1263 with MEM, 322 ± 229 with ketorolac 0.45% (significantly reduced, P < 0.01 vs bromfenac 0.09% and nepafenac 0.1%, 29,093 ± 14,295 with bromfenac 0.09% (significantly enlarged, P < 0.01 vs MEM and 47,322 ± 13,736 with nepafenac 0.1% (significantly enlarged, P < 0.01 vs MEM and vs

Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

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Justin D Mallet

Full Text Available Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD and pyrimidine (6-4 pyrimidone photoproducts (6-4PP. These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced.

Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals

OpenAIRE

Akihiro Higuchi; Hiroyoshi Inoue; Yoshio Kaneko; Erina Oonishi; Kazuo Tsubota

2016-01-01

The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy ...

Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals.

Science.gov (United States)

Higuchi, Akihiro; Inoue, Hiroyoshi; Kaneko, Yoshio; Oonishi, Erina; Tsubota, Kazuo

2016-11-11

The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy of Se-lactoferrin eye drops in the tobacco smoke exposure rat dry eye model and short-term rabbit dry eye model, although Diquas eye drops were only effective in the short-term rabbit dry eye model. These results indicate that Se-lactoferrin was useful in the oxidative stress-causing dry eye model. Se-lactoferrin was taken into corneal epithelium cells via lactoferrin receptors. We identified LRP1 as the lactoferrin receptor in the corneal epithelium involved in lactoferrin uptake. Se-lactoferrin eye drops did not irritate the ocular surface of rabbits. Se-lactoferrin was an excellent candidate for treatment of dry eye, reducing oxidative stress by a novel mechanism.

Epithelial cell polarity, stem cells and cancer

DEFF Research Database (Denmark)

Martin-Belmonte, Fernando; Perez-Moreno, Mirna

2011-01-01

, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

Spontaneous Corneal Hydrops in a Patient with a Corneal Ulcer

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Hatim Batawi

2016-01-01

Full Text Available Purpose: We report the case of a 77-year-old man with no history of keratoconus or other ectatic disorders who presented with corneal hydrops in the setting of a corneal ulcer. The risk factors, pathogenesis and treatment options of corneal hydrops are discussed. Method: This is an observational case report study. Results: A 77-year-old man presented with a 1-day history of severe pain, redness, mucous discharge and photophobia in the right eye. A slit-lamp examination of the right eye showed an area of focal corneal edema and protrusion. Within the area of edema and protrusion, there was an infiltrate with an overlying epithelial defect consistent with an infectious corneal ulcer. The Seidel test showed no leakage, so a clinical diagnosis of corneal hydrops associated with nonperforated corneal ulcer was made. With appropriate antibiotic treatment, the corneal ulcer and hydrops both resolved over a 1-month period. Conclusion: Corneal hydrops can occur in the setting of corneal infections.

A histological study of rabbit corneas after transepithelial corneal crosslinking using partial epithelial photoablation or ethanol treatment.

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Ozmen, Mehmet Cuneyt; Hondur, Ahmet; Yilmaz, Guldal; Bilgihan, Kamil; Hasanreisoglu, Berati

2014-01-01

To evaluate the histological changes after transepithelial corneal crosslinking (CXL) using partial thickness excimer laser ablation or epithelial ethanol application in an experimental rabbit study. Right eyes of twenty-four rabbits were studied. Four eyes received total epithelial debridement (group I). Four eyes received partial thickness epithelial ablation with excimer laser (group II). Twelve eyes were treated with different durations (30s and 60s) and concentrations (18% to 48%) of ethanol (group III). Riboflavin was applied for 30min intervals along with topical proparacaine drops with benzalkonium chloride, and 370 nm irradiation was performed for 30min, while riboflavin was instilled every 3min. Four eyes (group IV) received 48% ethanol for 30s without riboflavin and irradiation. Eyes were collected after 24h and examined histologically. All eyes in group I showed keratocyte loss in the superficial 300 µ of corneal storma. In group II, 1-4 layers of epithelium were preserved and no keratocyte loss occurred. In group III, CXL after treatment with ethanol up to 24% concentration and up to 60s revealed no keratocyte loss. CXL after treatment with 48% and higher ethanol concentrations yielded keratocyte loss in the superficial 200 µ to 300 µ of cornea. Incomplete excimer laser ablation of the epithelium or treatment with ethanol up to 24% concentration and up to 60s duration yielded no stromal keratocyte loss. To get the same histological appearance seen in epithelial debridement group, partial thickness excimer laser epithelial ablation or ethanol application is not adequate for transepithelial CXL.

A histological study of rabbit corneas after transepithelial corneal crosslinking using partial epithelial photoablation or ethanol treatment

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Mehmet Cuneyt Ozmen

2014-12-01

Full Text Available AIM: To evaluate the histological changes after transepithelial corneal crosslinking (CXL using partial thickness excimer laser ablation or epithelial ethanol application in an experimental rabbit study.METHODS: Right eyes of twenty-four rabbits were studied. Four eyes received total epithelial debridement (group I. Four eyes received partial thickness epithelial ablation with excimer laser (group II. Twelve eyes were treated with different durations (30s and 60s and concentrations (18% to 48% of ethanol (group III. Riboflavin was applied for 30min intervals along with topical proparacaine drops with benzalkonium chloride, and 370 nm irradiation was performed for 30min, while riboflavin was instilled every 3min. Four eyes (group IV received 48% ethanol for 30s without riboflavin and irradiation. Eyes were collected after 24h and examined histologically.RESULTS: All eyes in group I showed keratocyte loss in the superficial 300 µ of corneal storma. In group II, 1-4 layers of epithelium were preserved and no keratocyte loss occurred. In group III, CXL after treatment with ethanol up to 24% concentration and up to 60s revealed no keratocyte loss. CXL after treatment with 48% and higher ethanol concentrations yielded keratocyte loss in the superficial 200 µ to 300 µ of cornea.CONCLUSION: Incomplete excimer laser ablation of the epithelium or treatment with ethanol up to 24% concentration and up to 60s duration yielded no stromal keratocyte loss. To get the same histological appearance seen in epithelial debridement group, partial thickness excimer laser epithelial ablation or ethanol application is not adequate for transepithelial CXL.

Using corneal confocal microscopy to track changes in the corneal layers of dry eye patients after autologous serum treatment.

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Mahelkova, Gabriela; Jirsova, Katerina; Seidler Stangova, Petra; Palos, Michalis; Vesela, Viera; Fales, Ivan; Jiraskova, Nada; Dotrelova, Dagmar

2017-05-01

In vivo corneal confocal microscopy allows the examination of each layer of the cornea in detail and the identification of pathological changes at the cellular level. The purpose of this study was to identify the possible effects of a three-month treatment with autologous serum eye-drops in different corneal layers of patients with severe dry eye disease using corneal confocal microscopy. Twenty-six patients with dry eye disease were included in the study. Corneal fluorescein staining was performed. The corneas of the right eyes were examined using in vivo corneal confocal microscopy before and after a three-month treatment with autologous serum drops. The densities of superficial and basal epithelial cells, Langerhans cells, the keratocytes and activated keratocytes, the density of endothelial cells and the status of the sub-basal nerve plexus fibres were evaluated. A significant decrease in corneal fluorescein staining was found after the three-month autologous serum treatment (p = 0.0006). The basal epithelial cell density decreased significantly (p = 0.001), while the density of superficial epithelial cells did not change significantly (p = 0.473) nor did the number of Langerhans cells or activated keratocytes (p = 0.223; p = 0.307, respectively). There were no differences in the other corneal cell layers or in the status of the nerve fibres. The results demonstrate the ability of corneal confocal microscopy to evaluate an improvement in the basal epithelial cell layer of the cornea after autologous serum treatment in patients with dry eye disease. More studies with longer follow-up periods are needed to elucidate the suitability of corneal confocal microscopy to follow the effect of autologous serum treatment on nerve fibres or other corneal layers in dry eye disease patients. © 2016 Optometry Australia.

Ocular Surface Reconstruction with Cultivated Limbal Epithelial Cells in Limbal Stem Cell Deficiency: One-year Follow-up Results

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İsmet Durak

2012-05-01

Full Text Available Pur po se: To evaluate the 1-year follow-up results of cultivated limbal epithelial cell (CLEC transplantation in unilateral limbal stem cell deficiency (LSCD. Ma te ri al and Met hod: One-year follow-up results of five unilateral LSCD patients who had undergone CLEC transplantation were evaluated. Parameters for this evaluation were: fluorescein staining of ocular surface, corneal vascularization and status of epithelium with slit lamp, and visual acuity. 1.5-mm limbal biopsy was performed from the superior limbus of the healthy eyes, broke into two equal pieces, expanded on human amniotic membrane (hAM and inserts for 14 days until getting 20 mm in size. CLECs on hAMs were used directly, and cells on inserts were usedafter detachment procedure. The symblepharon and pannus tissues were removed, superficial keratectomy was performed. CLEC on hAMs were transplanted with the epithelial side up onto the bare corneal stroma, sutured to the conjunctiva with 10-0 nylon sutures. Free CLEC layer from insert was placed on hAM as a second layer, additional hAM was used as a protective layer all over other tissues. Re sults: Median age was 44.4 years (14-71. The etiology was chemical burn in all patients. Median duration of symptoms was 10 years (2-18, median follow-up period was 12.6 (12-12.5 months. Preoperative best corrected visual acuities (BCVA were light perception in three patients, counting fingers at 50 cm in one patient and 3/10 in one patient. Visions were improved in all patients. Postoperative BCVA 12 months after the surgery were between counting fingers at 3 meters to 6/10. There was a temporary hemorrhage between the two layers of hAMs in one patient at the early postoperative period. Peripheral corneal vascularization has occurred in three patients, in patient corneal vascularization has reached to the paracentral area. Dis cus si on: CLEC transplantation is an efficient treatment option for unilateral LSCD in mid-long term. (Turk J

Meta analysis of therapeutic effects of domestic deproteinized calf blood extract eye gel on corneal epithelial repair after laser refractive surgery

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Jian Zhang

2018-01-01

Full Text Available AIM: To systemically evaluate the clinical efficacy and safety of domestic deproteinized calf blood extract eye gel for corneal epithelial repair after laser refractive surgery. METHODS:We performed a comprehensive search via Pubmed, Embase, Cochrane Library, VIP Chinese Science and Technology Journal Database, CNKI and Wan Fang Chinese periodical Database for the randomized controlled trials(RCTsat home and abroad about effects of the domestic deproteinized calf blood extract eye gel for corneal epithelial repair after laser corneal refractive surgery with retrieval time from January 2007 to December 2016. According to the inclusion and exclusion criteria, 2 medical researchers independently screened documents, extracted data and evaluated the quality. Review Manager 5.3 software was used for Meta analysis. RESULTS: Seven RCTs involving 1 042 eyes, including 523 eyes in the treatment group and 519 eyes in the control group, were selected for this Meta-analysis. The results showed that the clinical efficacy in the treatment group was better than that in the control group(OR=1.81, 95%CI: 1.39～2.35; PWMD=-0.33, 95%CI: -0.45 to -0.21; PWMD=-1.26, 95%CI: -1.56 to -0.97; PCONCLUSION: The domestic deproteinized calf blood extract eye gel can relieve the patients' symptoms after laser refractive surgery, improve the corneal epithelial recovery and the efficiency of treatment. Due to the limited quality and quantity of the studies these conclusions should be further validated by more well-designed randomized double blind controlled trials.

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

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Yamamoto, Naoki; Kato, Yoshinao; Sato, Atsushi; Hiramatsu, Noriko; Yamashita, Hiromi; Ohkuma, Mahito; Miyachi, Ei-Ichi; Horiguchi, Masayuki; Hirano, Koji; Kojima, Hajime

2016-08-01

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

Keratocyte apoptosis and corneal antioxidant enzyme activities after refractive corneal surgery.

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Bilgihan, K; Bilgihan, A; Adiguzel, U; Sezer, C; Yis, O; Akyol, G; Hasanreisoglu, B

2002-01-01

Refractive corneal surgery induces keratocyte apoptosis and generates reactive oxygen radicals (ROS) in the cornea. The purpose of the present study is to evaluate the correlation between keratocyte apoptosis and corneal antioxidant enzyme activities after different refractive surgical procedures in rabbits. Rabbits were divided into six groups. All groups were compared with the control group (Group 1), after epithelial scraping (Group 2), epithelial scrape and photorefractive keratectomy (PRK) (traditional PRK: Group 3), transepithelial PRK (Group 4), creation of a corneal flap with microkeratome (Group 5) and laser-assisted in situ keratomileusis (LASIK, Group 6). Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay (to detect DNA fragmentation in situ) and light microscopy were used to detect apoptosis in rabbit eyes. Glutathione peroxidase (Gpx) and superoxide dismutase (SOD) activities of the corneal tissues were measured with spectrophotometric methods. Corneal Gpx and SOD activities decreased significantly in all groups when compared with the control group (P<0.05) and groups 2, 3 and 6 showed a significantly higher amount of keratocyte apoptosis (P<0.05). Not only a negative correlation was observed between corneal SOD activity and keratocyte apoptosis (cc: -0.3648) but Gpx activity also showed negative correlation with keratocyte apoptosis (cc: -0.3587). The present study illustrates the negative correlation between keratocyte apoptosis and corneal antioxidant enzyme activities. This finding suggests that ROS may be partly responsible for keratocyte apoptosis after refractive surgery.

Pirfenidone nanoparticles improve corneal wound healing and prevent scarring following alkali burn.

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Sushovan Chowdhury

Full Text Available To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn.Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide (PLGA nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry.Pirfenidone prevented (P<0.05 increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05 reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn.Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases.

IκB kinase β regulates epithelium migration during corneal wound healing.

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Liang Chen

2011-01-01

Full Text Available The IKKβ is known to regulate transcription factor NF-κB activation leading to inflammatory responses. Recent gene knockout studies have shown that IKKβ can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKKβ has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12(rtTA/rtTAt/tet-O-Cre/Ikkβ(F/F (Ikkβ(ΔCE/ΔCE mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F mice. Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi also showed that IKKβ was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKKβ was required for optimal activation of NF-κB and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKKβ-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKKβ in the control of epithelial cell motility and wound healing.

Microspectroscopy of spectral biomarkers associated with human corneal stem cells

OpenAIRE

Nakamura, Takahiro; Kelly, Jemma G.; Trevisan, J?lio; Cooper, Leanne J.; Bentley, Adam J.; Carmichael, Paul L.; Scott, Andrew D.; Cotte, Marine; Susini, Jean; Martin-Hirsch, Pierre L.; Kinoshita, Shigeru; Fullwood, Nigel J.; Martin, Francis L.

2010-01-01

Purpose Synchrotron-based radiation (SRS) Fourier-transform infrared (FTIR) microspectroscopy potentially provides novel biomarkers of the cell differentiation process. Because such imaging gives a ?biochemical-cell fingerprint? through a cell-sized aperture, we set out to determine whether distinguishing chemical entities associated with putative stem cells (SCs), transit-amplifying (TA) cells, or terminally-differentiated (TD) cells could be identified in human corneal epithelium. Methods D...

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

OpenAIRE

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell proper...

Plasticity between Epithelial and Mesenchymal States Unlinks EMT from Metastasis-Enhancing Stem Cell Capacity

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Evelyne Beerling

2016-03-01

Full Text Available Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells may adapt transient and reversible states. Here, we have tested the existence and role of epithelial-mesenchymal plasticity in metastasis of mammary tumors without artificially modifying EMT regulators. In these tumors, we found by intravital microscopy that the motile tumor cells have undergone EMT, while their epithelial counterparts were not migratory. Moreover, we found that epithelial-mesenchymal plasticity renders any EMT-induced stemness differences, as reported previously, irrelevant for metastatic outgrowth, because mesenchymal cells that arrive at secondary sites convert to the epithelial state within one or two divisions, thereby obtaining the same stem cell potential as their arrived epithelial counterparts. We conclude that epithelial-mesenchymal plasticity supports migration but additionally eliminates stemness-enhanced metastatic outgrowth differences.

Topical Drug Formulations for Prolonged Corneal Anesthesia

Science.gov (United States)

Wang, Liqiang; Shankarappa, Sahadev A.; Tong, Rong; Ciolino, Joseph B.; Tsui, Jonathan H.; Chiang, Homer H.; Kohane, Daniel S.

2013-01-01

Purpose Ocular local anesthetics (OLA’s) currently used in routine clinical practice for corneal anesthesia are short acting and their ability to delay corneal healing makes them unsuitable for long-term use. In this study, we examined the effect on the duration of corneal anesthesia of the site-1 sodium channel blocker tetrodotoxin (TTX), applied with either proparacaine or the chemical permeation enhancer OTAB. The effect of test solutions on corneal healing was also studied. Methods Solutions of TTX, proparacaine, and OTAB, singly or in combination were applied topically to the rat cornea. The blink response, an indirect measure of corneal sensitivity, was recorded using a Cochet-Bonnet esthesiometer, and the duration of corneal anesthesia calculated. The effect of test compounds on the rate of corneal epithelialization was studied in vivo following corneal debridement. Results Combination of TTX and proparacaine resulted in corneal anesthesia that was 8–10 times longer in duration than that from either drug administered alone, while OTAB did not prolong anesthesia. The rate of corneal healing was moderately delayed following co-administration of TTX and proparacaine. Conclusion Co-administration of TTX and proparacaine significantly prolonged corneal anesthesia but in view of delayed corneal re-epithelialization, caution is suggested in use of the combination. PMID:23615270

Effect of the combination of basic fibroblast growth factor and cysteine on corneal epithelial healing after photorefractive keratectomy in patients affected by myopia

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Alessandro Meduri

2014-01-01

Full Text Available Background: This study sought to evaluate the effect of basic fibroblast growth factor eye drops and cysteine oral supplements on corneal healing in patients treated with photorefractive keratectomy (PRK. Materials and Methods: One hundred and twenty patients treated bilaterally with PRK for myopia were enrolled at one of two eye centers (Clinica Santa Lucia, Bologna, Italy and Department of Ophthalmology, University of Magna Graecia, Catanzaro, Italy and were treated at the former center. Sixty patients included in the study group (Group 1 were treated postoperatively with topical basic fibroblast growth factor plus oral L-cysteine supplements, whereas 60 subjects included in the control group (Group 2 received basic fibroblast growth factor eye drops. We recorded the rate of corneal re-epithelialization and patients were followed-up every 30 days for 6 months. Statistical analyses were performed on the collected data. Results: The eyes in Group 1 demonstrated complete re-epithelialization at Day 5, whereas the eyes in Group 2 achieved this status on Day 6. No side-effects were reported. Conclusions : Patients treated with basic fibroblast growth factor eye drops and L-cysteine oral supplements benefit from more rapid corneal re-epithelialization. In human eyes, this combination treatment appeared to be safe and effective in accelerating corneal surfacing after surgery. Financial Disclosure: No author has any financial or proprietary interest in any material or method used in this study. Trial Registration: Current Controlled Trials ISRCTN73824458.

Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

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Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

2008-01-01

The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET.

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Shalom-Feuerstein, Ruby; Serror, Laura; Aberdam, Edith; Müller, Franz-Josef; van Bokhoven, Hans; Wiman, Klas G; Zhou, Huiqing; Aberdam, Daniel; Petit, Isabelle

2013-02-05

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

Science.gov (United States)

Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

2016-02-01

To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

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Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

2015-01-01

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. © 2014 AlphaMed Press.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

OpenAIRE

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

2007-01-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipa...

Spontaneous Healing of Corneal Perforation after Temporary Discontinuation of Erlotinib Treatment

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Naoyuki Morishige

2014-01-01

Full Text Available Purpose: To report a case of corneal perforation associated with oral administration of erlotinib and its spontaneous healing after temporary discontinuation of drug treatment. Case Report: A 65-year-old man with metastatic lung cancer was treated with erlotinib (150 mg/day, a specific tyrosine kinase inhibitor of the epidermal growth factor receptor. He was referred to our corneal service for the treatment of bilateral corneal disorders, diagnosed with mild aqueous-deficient dry eye, and treated by insertion of punctal plugs. His corneal epithelial disorders initially improved, but subsequently worsened, as manifested by the development of bilateral corneal ulceration with corneal perforation in the right eye. The oral administration of erlotinib was interrupted in preparation for tectonic keratoplasty, but 2 days later the corneal perforation of the right eye and the bilateral epithelial defects had healed spontaneously. Treatment with erlotinib was resumed at half the initial dose, and the cornea of both eyes has remained apparently healthy. Discussion: Erlotinib may be secreted into tear fluid and thereby adversely affect the corneal epithelium. The development of corneal epithelial disorders in patients receiving this drug may be reversed by reducing its dose.

Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells

Science.gov (United States)

2010-01-01

inflammatory cytokine release in corneal epithelium through MAPK signaling. J. Cell Physiol. 213, 730e739. Zhang, J., Li , H., Wang, J., Dong, Z., Mian ...Kang, S.S., Li , T., Xu, D., Reinach, P.S., Lu, L., 2000. Inhibitory effect of PGE2 on EGF- induced MAP kinase activity and rabbit corneal epithelial

Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model.

Science.gov (United States)

Tanaka, Yohei; Nakayama, Jun

2016-01-01

Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000-1,800 nm wavelengths and excluded 1,400-1,500 nm wavelengths. A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm(2) irradiation (Psolar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in turn eye damage.

Bilateral congenital corneal keloids and anterior segment mesenchymal dysgenesis in a case of Rubinstein-Taybi syndrome.

Science.gov (United States)

Rao, Srinivas K; Fan, Dorothy S P; Pang, C P; Li, Winnie W Y; Ng, Joan S K; Good, William V; Lam, Dennis S C

2002-01-01

To report the unusual association of bilateral corneal keloids and anterior segment mesenchymal dysgenesis in a child with Rubinstein-Taybi syndrome. Case report of a 2-year-old boy. Excision of the epicorneal mass in the right eye was followed by recurrence of the lesion. Multiple penetrating keratoplasties were unsuccessful in reconstructing the anterior segment because of recurrent corneal epithelial breakdown, suggesting limbal stem cell insufficiency. Histopathology and electron microscopy of the excised mass lesion showed features typical of a corneal keloid: thickened keratinized epithelium, absent Bowman's layer, and fibrovascular hyperplasia, with haphazard orientation of the collagen lamellae. Ultrasound biomicroscopy and intraoperative findings suggested a diagnosis of Peter anomaly, but genetic analysis did not show a PAX6 mutation. The findings in our patient add to the spectrum of ocular changes described in Rubinstein-Taybi syndrome and confirm earlier reports of poor ocular prognosis in corneal keloids and Rubinstein-Taybi syndrome.

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

Science.gov (United States)

Turner, Helen C; Budak, Murat T; Akinci, M A Murat; Wolosin, J Mario

2007-05-01

To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that

Corneal Toxicity Following Exposure to Asclepias Tuberosa

OpenAIRE

Mikkelsen, Lauge Hjorth; Hamoudi, Hassan; G?l, Cigdem Altuntas; Heegaard, Steffen

2017-01-01

PURPOSE: To present a case of corneal toxicity following exposure to milky plant latex from Asclepias tuberosa.METHODS: A 70-year-old female presented with blurred vision and pain in her left eye after handling an Ascepias tuberosa. Clinical examination revealed a corneal stromal oedema with small epithelial defects. The corneal endothelium was intact and folds in Descemets membrane were observed. The oedema was treated with chloramphenicol, dexamethasone and scopolamine.RESULTS: The corneal ...

A Comparative Study of the Therapeutic Potential of Mesenchymal Stem Cells and Limbal Epithelial Stem Cells for Ocular Surface Reconstruction

Czech Academy of Sciences Publication Activity Database

Holáň, Vladimír; Trošan, Peter; Čejka, Čestmír; Javorková, Eliška; Zajícová, Alena; Heřmánková, Barbora; Chudíčková, Milada; Čejková, Jitka

2015-01-01

Roč. 4, č. 9 (2015), s. 1052-1063 ISSN 2157-6564 R&D Projects: GA ČR(CZ) GA14-12580S; GA MZd NT14102; GA MŠk(CZ) LO1309; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378041 Keywords : limbal stem cells * mesenchymal stem cells * alkali-injured ocular surface * corneal regeneration * stem cell-based therapy Subject RIV: FF - HEENT, Dentistry Impact factor: 4.247, year: 2015

Glycosaminoglycans are involved in pathogen adherence to corneal epithelial cells differently for Gram-positive and Gram-negative bacteria

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Beatriz García

2016-11-01

Full Text Available The epithelium of the cornea is continuously exposed to pathogens, and adhesion to epithelial cells is regarded as an essential first step in bacterial pathogenesis. In this article, the involvement of glycosaminoglycans in the adhesion of various pathogenic bacteria to corneal epithelial cells is analyzed. All microorganisms use glycosaminoglycans as receptors, but arranged in different patterns depending on the Gram-type of the bacterium. The heparan sulfate chains of syndecans are the main receptors, though other molecular species also seem to be involved, particularly in Gram-negative bacteria. Adherence is inhibited differentially by peptides, including heparin binding sequences, indicating the participation of various groups of Gram-positive and -negative adhesins. The length of the saccharides produces a major effect, and low molecular weight chains inhibit the binding of Gram-negative microorganisms but increase the adherence of Gram-positives. Pathogen adhesion appears to occur preferentially through sulfated domains, and is very dependent on N- and 6-O-sulfation of the glucosamine residue and, to a lesser extent, 2-O sulfation of uronic acid. These data show the differential use of corneal receptors, which could facilitate the development of new anti-infective strategies.

Overexpression of microRNA-194 suppresses the epithelial-mesenchymal transition in targeting stem cell transcription factor Sox3 in endometrial carcinoma stem cells.

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Gong, Baolan; Yue, Yan; Wang, Renxiao; Zhang, Yi; Jin, Quanfang; Zhou, Xi

2017-06-01

The epithelial-mesenchymal transition is the key process driving cancer metastasis. MicroRNA-194 inhibits epithelial-mesenchymal transition in several cancers and its downregulation indicates a poor prognosis in human endometrial carcinoma. Self-renewal factor Sox3 induces epithelial-mesenchymal transition at gastrulation and is also involved epithelial-mesenchymal transition in several cancers. We intended to determine the roles of Sox3 in inducing epithelial-mesenchymal transition in endometrial cancer stem cells and the possible role of microRNA-194 in controlling Sox3 expression. Firstly, we found that Sox3 and microRNA-194 expressions were associated with the status of endometrial cancer stem cells in a panel of endometrial carcinoma tissue, the CD133+ cell was higher in tumorsphere than in differentiated cells, and overexpression of microRNA-194 would decrease CD133+ cell expression. Silencing of Sox3 in endometrial cancer stem cell upregulated the epithelial marker E-cadherin, downregulated the mesenchymal marker vimentin, and significantly reduced cell invasion in vitro; overexpression of Sox3 reversed these phenotypes. Furthermore, we discovered that the expression of Sox3 was suppressed by microRNA-194 through direct binding to the Sox3 3'-untranslated region. Ectopic expression of microRNA-194 in endometrial cancer stem cells induced a mesenchymal-epithelial transition by restoring E-cadherin expression, decreasing vimentin expression, and inhibiting cell invasion in vitro. Moreover, overexpression of microRNA-194 inhibited endometrial cancer stem cell invasion or metastasis in vivo by injection of adenovirus microRNA-194. These findings demonstrate the novel mechanism by which Sox3 contributes to endometrial cancer stem cell invasion and suggest that repression of Sox3 by microRNA-194 may have therapeutic potential to suppress endometrial carcinoma metastasis. The cancer stem cell marker, CD133, might be the surface marker of endometrial cancer stem

Instillation of Sericin Enhances Corneal Wound Healing through the ERK Pathway in Rat Debrided Corneal Epithelium

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Noriaki Nagai

2018-04-01

Full Text Available Sericin is a major constituent of silk produced by silkworms. We previously found that the instillation of sericin enhanced the proliferation of corneal epithelial cells, and acted to promote corneal wound healing in both normal and diabetic model rats. However, the mechanisms by which sericin promotes the proliferation of corneal cells have not been established. In this study, we investigated the effects of sericin on Akt and ERK activation in a human corneal epithelial cell line (HCE-T cells and rat debrided corneal epithelium. Although Akt phosphorylation was not detected following the treatment of HCE-T cells with sericin, ERK1/2 phosphorylation was enhanced. The growth of HCE-T cells treated with sericin was significantly increased, with the cell growth of sericin-treated HCE-T cells being 1.7-fold higher in comparison with vehicle-treated HCE-T cells. On the other hand, both of an ERK inhibitor U0126 (non-specific specific inhibitor and SCH772984 (specific inhibitor attenuated the enhanced cell growth by sericin, and the growth level in the case of co-treatment with sericin and ERK1/2 inhibitor was similar to that of cells treated with ERK1/2 inhibitor alone. In an in vivo study using rat debrided corneal epithelium, the corneal wound healing rate was enhanced by the instillation of sericin, and this enhancement was also attenuated by the instillation of U0126. In addition, the corneal wound healing rate in rats co-instilled with sericin and U0126 was similar to that following the instillation of U0126 alone. In conclusion, we found that the instillation of sericin enhanced cell proliferation via the activation of the MAPK/ERK pathway, resulting in the promotion of corneal wound healing in rat eyes. These findings provide significant information for designing further studies to develop potent corneal wound-healing drugs.

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

DEFF Research Database (Denmark)

Bath, Chris; Yang, Sufang; Muttuvelu, Danson

2013-01-01

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth......, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE......, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET)....

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

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Lai JY

2013-10-01

Full Text Available Jui-Yang Lai,1–3 David Hui-Kang Ma4,5 1Institute of Biochemical and Biomedical Engineering, 2Biomedical Engineering Research Center, 3Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; 4Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 5Department of Chinese Medicine, Chang Gung University, Taoyuan, Taiwan Abstract: Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA cross-linked amniotic membrane (AM on limbal epithelial cell (LEC cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous

Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.

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Satoru Yoshida

Full Text Available BACKGROUND: Application of induced pluripotent stem (iPS cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. METHODOLOGY/PRINCIPAL FINDINGS: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. CONCLUSIONS/SIGNIFICANCE: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.

RECURRENT CORNEAL EROSION SYNDROME (a review

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S. V. Trufanov

2015-01-01

Full Text Available Recurrent corneal erosion (RCE syndrome is characterized by episodes of recurrent spontaneous epithelial defects. Main clinical symptoms (pain, redness, photophobia, lacrimation occurred at night. Corneal lesions revealed by slit lamp exam vary depending on the presence of corneal epithelium raise, epithelial microcysts or epithelial erosions, stromal infiltrates and opacities. Microtraumas, anterior corneal dystrophies, and herpesvirus give rise to RCE. Other causes or factors which increase the risk of RCE syndrome include meibomian gland dysfunction, keratoconjunctivitis sicca, diabetes, and post-LASIK conditions. Basal membrane abnormalities and instability of epithelial adhesion to stroma play a key role in RCE pathogenesis. Ultrastructural changes in RCE include abnormalities of basal epithelial cells and epithelial basal membrane, absence or deficiency of semi-desmosomes, loss of anchor fibrils. Increase in matrix metalloproteinases and collagenases which contribute to basal membrane destruction results in recurrent erosions and further development of abnormal basal membrane. The goals of RCE therapy are to reduce pain (in acute stage, to stimulate re-epithelization, and to restore «adhesion complex» of basal membrane. In most cases, RCE responds to simple conservative treatment that includes lubricants, healing agents, and eye patches. RCEs that are resistant to simple treatment, require complex approach. Non-invasive methods include long-term contact lens use, instillations of autologous serum (eye drops, injections of botulinum toxin (induces ptosis, antiviral agent use or oral intake of metalloproteinase inhibitors. Cell membrane stabilizers, i.e., antioxidants, should be included into treatment approaches as well. Antioxidant effect of Emoxipine promotes tissue reparation due to the prevention of cell membrane lipid peroxidation as well as due to its anti-hypoxic, angioprotective, and antiplatelet effects. If conservative therapy

Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model

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Tanaka Y

2016-08-01

Full Text Available Yohei Tanaka,1,2 Jun Nakayama2 1Department of Plastic Surgery, Clinica Tanaka Plastic, Reconstructive Surgery and Anti-aging Center, 2Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan Background and objective: Humans are increasingly exposed to near-infrared (NIR radiation from both natural (eg, solar and artificial (eg, electrical appliances sources. Although the biological effects of sun and ultraviolet (UV exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues.Materials and methods: DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C. The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths.Results: A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05.Conclusion: We found that NIR irradiation induced the

Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants

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Kai-Hung Wang

2016-01-01

Full Text Available We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40 of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

Conjunctival-limbal autograft in total unilateral limbal stem cell deficiency

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Petra Schollmayer

2017-09-01

Full Text Available Background: Corneal epithelium is renewed by stem cells (SC that reside at the corneal limbus. Reduced number of SC or their abnormal function lead to the ocular surface disease called limbal stem cell deficiency (LSCD, characterized by corneal conjunctivalization, vascularization, persistent epithelial defects, chronic inflammation, and loss of vision. In a case of total unilateral LSCD, autologous transplantation of limbal epithelial stem cells (LESC from the healthy eye is needed. We describe the surgical technique of choice for autologous limbal transplantation, called conjunctival limbal autograft (CLAU that we combined with amniotic membrane (AM use. We present the results of CLAU in three patients with total unilateral LSCD due to chemical injury.Methods: Autologous limbal transplantation CLAU begins with the removal of fibrovascular pannus from the diseased corneal surface and the harvesting of two conjunctival-limbal grafts from the healthy eye. The grafts are then transplanted on to the limbal area of the recipient eye. AM is used as a patch to cover the denuded cornea and limbal grafts, as well as a barrier preventing the conjunctival epithelium from encroaching on to the temporal and nasal side of the corneal surface. In the donor eye, AM is used to cover the donor sites. CLAU with the use of AM was performed in 3 patients with unilateral LSCD due to chemical eye injury. In one patient limbal transplantation was combined with symblepharon lysis for entropium repair. In all cases AM was removed 3–6 days postoperatively to assess the growth of new epithelium from the limbal grafts. In all patients the ocular surface was covered with another AM until the cornea was completely epithelized and the new epithelium stable. In one patient the corneal regrafting and cataract removal was performed subsequently.Results: CLAU was successful in 2 patients and partially successful in 1 patient during the follow up. In all cases the growth of new

Healed corneal ulcer with keloid formation.

Science.gov (United States)

Alkatan, Hind M; Al-Arfaj, Khalid M; Hantera, Mohammed; Al-Kharashi, Soliman

2012-04-01

We are reporting a 34-year-old Arabic white female patient who presented with a white mass covering her left cornea following multiple ocular surgeries and healed corneal ulcer. The lesion obscured further view of the iris, pupil and lens. The patient underwent penetrating keratoplasty and the histopathologic study of the left corneal button showed epithelial hyperplasia, absent Bowman's layer and subepithelial fibrovascular proliferation. The histopathologic appearance was suggestive of a corneal keloid which was supported by further ultrastructural study. The corneal graft remained clear 6 months after surgery and the patient was satisfied with the visual outcome. Penetrating keratoplasty may be an effective surgical option for corneal keloids in young adult patients.

Oral epithelial stem cells – implications in normal development and cancer metastasis

Science.gov (United States)

Papagerakis, Silvana; Pannone, Giuseppe; Zheng, Li; About, Imad; Taqi, Nawar; Nguyen, Nghia P.T.; Matossian, Margarite; McAlpin, Blake; Santoro, Angela; McHugh, Jonathan; Prince, Mark E.; Papagerakis, Petros

2014-01-01

Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination. PMID:24803391

TNF-α promotes cell survival through stimulation of K+ channel and NFκB activity in corneal epithelial cells

International Nuclear Information System (INIS)

Wang Ling; Reinach, Peter; Lu, Luo

2005-01-01

Tumor necrosis factor (TNF-α) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-α also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-α stimulation induced activation of a voltage-gated K + channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-α on downstream events included NFκB nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-α induced increases in p21 expression resulting in partial cell cycle attenuation in the G 1 phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-α-induced K + channel activity effectively prevented NFκB nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-α. In conclusion, TNF-α promotes survival of HCE cells through sequential stimulation of K + channel and NFκB activities. This response to TNF-α is dependent on stimulating K + channel activity because following suppression of K + channel activity TNF-α failed to activate NFκB nuclear translocation and binding to nuclear DNA

Corneal wound healing is compromised by immunoproteasome deficiency.

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Deborah A Ferrington

Full Text Available Recent studies have revealed roles for immunoproteasome in regulating cell processes essential for maintaining homeostasis and in responding to stress and injury. The current study investigates how the absence of immunoproteasome affects the corneal epithelium under normal and stressed conditions by comparing corneas from wildtype (WT mice and those deficient in two immunoproteasome catalytic subunits (lmp7(-/-/mecl-1(-/-, L7M1. Immunoproteasome expression was confirmed in WT epithelial cells and in cells of the immune system that were present in the cornea. More apoptotic cells were found in both corneal explant cultures and uninjured corneas of L7M1 compared to WT mice. Following mechanical debridement, L7M1 corneas displayed delayed wound healing, including delayed re-epithelialization and re-establishment of the epithelial barrier, as well as altered inflammatory cytokine production compared to WT mice. These results suggest that immunoproteasome plays an important role in corneal homeostasis and wound healing.

C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

Science.gov (United States)

Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

2010-08-01

In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Valgardur Sigurdsson

Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

Science.gov (United States)

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Transcriptional profiling of putative human epithelial stem cells

Directory of Open Access Journals (Sweden)

Koçer Salih S

2008-07-01

may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.

Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components

OpenAIRE

Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun

2011-01-01

Purpose To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. Methods The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were...

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

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Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Effects of topical vitamin E on corneal superoxide dismutase, glutathione peroxidase activities and polymorphonuclear leucocyte infiltration after photorefractive keratectomy.

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Bilgihan, Ayse; Bilgihan, Kamil; Yis, Ozgür; Sezer, Cem; Akyol, Gülen; Hasanreisoglu, Berati

2003-04-01

Photorefractive keratectomy (PRK) induces free radical formation and polymorphonuclear (PMN) cell infiltration in the cornea. Vitamin E is a free radical scavenger and protects the cells from reactive oxygen species. We investigated the effects of topical vitamin E on corneal PMN cell infiltration and corneal antioxidant enzyme activities after PRK. We studied four groups, each consisting of seven eyes. Group 1 were control eyes. In group 2 the corneal epithelium was removed by a blunt spatula (epithelial scrape). In group 3, corneal photoablation (59 micro m, 5 dioptres) was performed after epithelial removal (traditional PRK). In group 4 we tested the effects of topical Vitamin E after traditional PRK. Corneal tissues were removed and studied with enzymatic analysis (measurement of corneal superoxide dismutase and glutathione peroxidase activities) and histologically. Stromal PMN leucocyte counts were significantly higher after mechanical epithelial removal and traditional PRK (p < 0.05). Corneal superoxide dismutase and glutathione peroxidase activities decreased significantly after mechanical epithelial removal and traditional PRK (p < 0.05). In group 4, treated with vitamin E, corneal superoxide dismutase activity did not differ significantly from that in the medically non-treated groups, nor did corneal PMN cell infiltration after traditional PRK. The reduction of corneal glutathione peroxidase activity after PRK was reduced significantly after topical vitamin E treatment. Topical vitamin E treatment may be useful for reducing the harmful effects of reactive oxygen radical after epithelial scraping and PRK in that it increases corneal glutathione peroxidase activity.

Mesenchymal Stromal Cell-Derived Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Acquisition of Epithelial Stem-Like Cell Properties in Ameloblastoma Epithelial Cells.

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Jiang, Chunmiao; Zhang, Qunzhou; Shanti, Rabie M; Shi, Shihong; Chang, Ting-Han; Carrasco, Lee; Alawi, Faizan; Le, Anh D

2017-09-01

Epithelial-mesenchymal transition (EMT), a biological process associated with cancer stem-like or cancer-initiating cell formation, contributes to the invasiveness, metastasis, drug resistance, and recurrence of the malignant tumors; it remains to be determined whether similar processes contribute to the pathogenesis and progression of ameloblastoma (AM), a benign but locally invasive odontogenic neoplasm. Here, we demonstrated that EMT- and stem cell-related genes were expressed in the epithelial islands of the most common histologic variant subtype, the follicular AM. Our results revealed elevated interleukin (IL)-6 signals that were differentially expressed in the stromal compartment of the follicular AM. To explore the stromal effect on tumor pathogenesis, we isolated and characterized both mesenchymal stromal cells (AM-MSCs) and epithelial cells (AM-EpiCs) from follicular AM and demonstrated that, in in vitro culture, AM-MSCs secreted a significantly higher level of IL-6 as compared to the counterpart AM-EpiCs. Furthermore, both in vitro and in vivo studies revealed that exogenous and AM-MSC-derived IL-6 induced the expression of EMT- and stem cell-related genes in AM-EpiCs, whereas such effects were significantly abrogated either by a specific inhibitor of STAT3 or ERK1/2, or by knockdown of Slug gene expression. These findings suggest that AM-MSC-derived IL-6 promotes tumor-stem like cell formation by inducing EMT process in AM-EpiCs through STAT3 and ERK1/2-mediated signaling pathways, implying a role in the etiology and progression of the benign but locally invasive neoplasm. Stem Cells 2017;35:2083-2094. © 2017 AlphaMed Press.

Corneal Toxicity Following Exposure to Asclepias Tuberosa

DEFF Research Database (Denmark)

Mikkelsen, Lauge Hjorth; Hamoudi, Hassan; Gül, Cigdem Altuntas

2017-01-01

PURPOSE: To present a case of corneal toxicity following exposure to milky plant latex from Asclepias tuberosa. METHODS: A 70-year-old female presented with blurred vision and pain in her left eye after handling an Ascepias tuberosa. Clinical examination revealed a corneal stromal oedema with small...... epithelial defects. The corneal endothelium was intact and folds in Descemets membrane were observed. The oedema was treated with chloramphenicol, dexamethasone and scopolamine. RESULTS: The corneal oedema had appeared after corneal exposure to the plant, Asclepias tuberosa, whose latex contains cardenolides...... that inhibit the Na+/ K+-ATPase in the corneal endothelium. The oedema resolved after 96 hours. After nine months the best corrected visual acuity was 20/20. CONCLUSION: Corneal toxicity has previously been reported for plants of the Asclepias family. This is a rare case describing severe corneal toxicity...

Correlation between the histological features of corneal surface pannus following ocular surface burns and the final outcome of cultivated limbal epithelial transplantation.

Science.gov (United States)

Sati, Alok; Basu, Sayan; Sangwan, Virender S; Vemuganti, Geeta K

2015-04-01

To report the influence of histological features of corneal surface pannus following ocular surface burn on the outcome of cultivated limbal epithelial transplantation (CLET). On retrospectively reviewing the medical records of the patients who underwent autologous CLET from April 2002 to June 2012 at L V Prasad Eye Institute, Hyderabad, India, we could trace the histological reports in only 90 records. These 90 records, besides clinical parameters, were reviewed for the influence of various histological features on the final outcome of CLET. The histological features include epithelial hyperplasia (21.1%), surface ulceration (2.2%), goblet cells (62.2%), squamous metaplasia (11.1%), active fibrosis (31.1%), severe inflammation (8.9%), multinucleated giant cells (3.3%), stromal calcification (8.9%) and active proliferating vessels (5.6%). Among these histological features, patients with either hyperplasia or calcification in their excised corneal pannus show an unfavourable outcome compared with patients without hyperplasia (p=0.003) or calcification (p=0.018). A similar unfavourable outcome was not seen with other histological features and various clinical parameters. Presence of either calcific deposits or hyperplasia in the excised corneal pannus provides poor prognostication; hence, a proper counselling of such patients is mandatory along with a close follow-up. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

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Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

2014-01-01

Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

Construction of Anterior Hemi-Corneal Equivalents Using Nontransfected Human Corneal Cells and Transplantation in Dog Models.

Science.gov (United States)

Xu, Bin; Song, Zhan; Fan, Tingjun

2017-11-01

Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells.

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Dimitrios Karamichos

Full Text Available Human corneal fibroblasts (HCF and corneal stromal stem cells (CSSC each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7. Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.

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Makiko Nakahara

Full Text Available Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs, even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM obtained from human bone marrow-derived mesenchymal stem cells (MSCs (MSC-CM for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na(+/K(+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27(Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary

Ultraviolet induced lysosome activity in corneal epithelium

International Nuclear Information System (INIS)

Cullen, A.P.

1980-01-01

A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm -2 to 10.000 Jm -2 and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm -2 and lens threshold (Hsub(L)) was 7.500 Jm -2 . The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared. (orig.) [de

Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

NARCIS (Netherlands)

P. Aparicio-Domingo (Patricia); M. Romera Hernández (Mónica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

2015-01-01

textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

Corneal Toxicity Following Exposure to Asclepias Tuberosa.

Science.gov (United States)

Mikkelsen, Lauge Hjorth; Hamoudi, Hassan; Gül, Cigdem Altuntas; Heegaard, Steffen

2017-01-01

To present a case of corneal toxicity following exposure to milky plant latex from Asclepias tuberosa. A 70-year-old female presented with blurred vision and pain in her left eye after handling an Ascepias tuberosa . Clinical examination revealed a corneal stromal oedema with small epithelial defects. The corneal endothelium was intact and folds in Descemets membrane were observed. The oedema was treated with chloramphenicol, dexamethasone and scopolamine. The corneal oedema had appeared after corneal exposure to the plant, Asclepias tuberosa , whose latex contains cardenolides that inhibit the Na + / K + -ATPase in the corneal endothelium. The oedema resolved after 96 hours. After nine months the best corrected visual acuity was 20/20. Corneal toxicity has previously been reported for plants of the Asclepias family. This is a rare case describing severe corneal toxicity caused by exposure to latex from Asclepias tuberosa . Handling of plants of the Asclepias family should be kept as a differential diagnosis in cases of acute corneal toxicity.

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

International Nuclear Information System (INIS)

Li Hongzhen; Zhou Jianjun; Miki, Jun; Furusato, Bungo; Gu Yongpeng; Srivastava, Shiv; McLeod, David G.; Vogel, Jonathan C.; Rhim, Johng S.

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin α2β1 hi and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 μg/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation

Safety of Cultivated Limbal Epithelial Stem Cell Transplantation for Human Corneal Regeneration

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J. Behaegel

2017-01-01

Full Text Available Ex vivo cultivated limbal stem cell transplantation is a promising technique for the treatment of limbal stem cell deficiency. While the results of the clinical trials have been extensively reported since the introduction of the technique in 1997, little has been reported regarding the potential health risks associated with production processes and transplantation techniques. Culture procedures require the use of animal and/or human-derived products, which carry the potential of introducing toxic or infectious agents through contamination with known or unknown additives. Protocols vary widely, and the risks depend on the local institutional methods. Good manufacturing practice and xeno-free culture protocols could reduce potential health risks but are not yet a common practice worldwide. In this review, we focus on the safety of both autologous- and allogeneic-cultivated limbal stem cell transplantation, with respect to culture processes, surgical approaches, and postoperative strategies.

Two cases of corneal perforation after oral administration of nonsteroidal anti-inflammatory drugs: oral NSAID-induced corneal damage.

Science.gov (United States)

Masuda, Ikuya; Matsuo, Toshihiko; Okamoto, Kazuo; Matsushita, Kyoko; Ohtsuki, Hiroshi

2010-01-01

To report 2 cases of corneal perforation associated with the use of oral nonsteroidal anti-inflammatory drugs (NSAIDs). In a 62-year-old woman and a 79-year-old woman, corneal perforation occurred after 7 days and 5 months of oral NSAIDs administration, respectively. After NSAIDs were discontinued, the cornea epithelialized and the anterior chamber formed within 14 and 10 days, respectively. It is well known that topical NSAIDs cause corneal perforation. Observations in the present cases suggest that the oral administration of NSAIDs may also cause corneal damage, and hence, medical professionals should consider the risk of damage to the cornea when administering these drugs orally.

Research on mouse model of grade II corneal alkali burn

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Jun-Qiang Bai

2016-04-01

Full Text Available AIM: To choose appropriate concentration of sodium hydroxide (NaOH solution to establish a stable and consistent corneal alkali burn mouse model in grade II. METHODS: The mice (n=60 were randomly divided into four groups and 15 mice each group. Corneal alkali burns were induced by placing circle filter paper soaked with NaOH solutions on the right central cornea for 30s. The concentrations of NaOH solutions of groups A, B, C, and D were 0.1 mol/L, 0.15 mol/L , 0.2 mol/L, and 1.0 mol/L respectively. Then these corneas were irrigated with 20 mL physiological saline (0.9% NaCl. On day 7 postburn, slit lamp microscope was used to observe corneal opacity, corneal epithelial sodium fluorescein staining positive rate, incidence of corneal ulcer and corneal neovascularization, meanwhile pictures of the anterior eyes were taken. Cirrus spectral domain optical coherence tomography was used to scan cornea to observe corneal epithelial defect and corneal ulcer. RESULTS: Corneal opacity scores ( were not significantly different between the group A and group B (P=0.097. Incidence of corneal ulcer in group B was significantly higher than that in group A (P=0.035. Incidence of corneal ulcer and perforation rate in group B was lower than that in group C. Group C and D had corneal neovascularization, and incidence of corneal neovascularization in group D was significantly higher than that in group C (P=0.000. CONCLUSION: Using 0.15 mol/L NaOH can establish grade II mouse model of corneal alkali burns.

Persistent Epithelial Defects and Corneal Opacity After Collagen Cross-Linking With Substitution of Dextran (T-500) With Dextran Sulfate in Compounded Topical Riboflavin.

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Höllhumer, Roland; Watson, Stephanie; Beckingsale, Peter

2017-03-01

Collagen cross-linking (CXL) is a commonly performed procedure to prevent the progression of keratoconus. Riboflavin is an essential part of the procedure, which facilitates both the cross-linking process and protection of intraocular structures. Dextran can be added to riboflavin to create an isotonic solution. This case report highlights the importance of compounding riboflavin with the correct dextran solution. A retrospective case series. Six eyes of 4 male patients with keratoconus aged from 20 to 38 years underwent CXL with substitution of 20% dextran (T-500) with 20% dextran sulfate in a compounded riboflavin 0.1% solution. Postoperatively, persistent corneal epithelial defects, stromal haze, and then scarring occurred. Corneal transplantation was performed for visual rehabilitation but was complicated by graft rejection followed by failure (n = 1 eye), dehiscence (n = 4), cataract (n = 2), post-laser ablation haze (n = 1), and steroid-induced glaucoma (n = 2). The visual outcome was dextran (T-500) with dextran sulfate in riboflavin solutions during CXL results in loss of vision from permanent corneal opacity. Residual host changes may compromise the results of corneal transplantation.

Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

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Le-Bel, Gaëtan; Ghio, Sergio Cortez; Larouche, Danielle; Germain, Lucie; Guérin, Sylvain L

2018-05-27

Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

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Loredana Alberti

Full Text Available Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites or CTCFL (CTCF-like is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1 and cancer stem cell markers (ABCG2, CD44 and ALDH1 genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7. Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

Ultraviolet induced lysosome activity in corneal epithelium

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Cullen, A.P.

1980-01-01

A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm/sup -2/ to 10.000 Jm/sup -2/ and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm/sup -2/ and lens threshold (Hsub(L)) was 7.500 Jm/sup -2/. The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared.

Healed corneal ulcer with keloid formation

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Alkatan, Hind M.; Al-Arfaj, Khalid M.; Hantera, Mohammed; Al-Kharashi, Soliman

2012-01-01

We are reporting a 34-year-old Arabic white female patient who presented with a white mass covering her left cornea following multiple ocular surgeries and healed corneal ulcer. The lesion obscured further view of the iris, pupil and lens. The patient underwent penetrating keratoplasty and the histopathologic study of the left corneal button showed epithelial hyperplasia, absent Bowman’s layer and subepithelial fibrovascular proliferation. The histopathologic appearance was suggestive of a co...

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

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Lai, Jui-Yang; Ma, David Hui-Kang

2013-01-01

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

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Morishige, Naoyuki; Ko, Ji-Ae; Morita, Yukiko; Nishida, Teruo

2010-01-01

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of

Preserved and unpreserved 12 anti-allergic ophthalmic solutions and ocular surface toxicity: in vitro assessment in four cultured corneal and conjunctival epithelial cell lines.

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Ayaki, Masahiko; Iwasawa, Atsuo; Yaguchi, Shigeo; Koide, Ryohei

2010-12-01

In the present study, we evaluated the cytotoxicity of anti-allergic ophthalmic solutions in cultured corneal and conjunctival cells, namely SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). The viability of cell cultures was determined following the exposure of cells to 12 commercially available anti-allergic ophthalmic solutions for varying exposure times and at various dilutions using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of different drugs. Based on CVS data, the order of cell viability after exposure to the drugs was Zepelin ≥ Tramelas PF ≥ Cumorol PF ≥ Ketotifen PF ≥ Eyevinal = Fumarton ≥ Cumorol > Intal ≥ Rizaben ≥ Tramelas ≥ Patanol Livostin. In conclusion, cell viability was mostly affected by the concentration of benzalkonium chloride rather than the active component and/or the anti-allergic action of the drug. The CVS was useful in comparing the toxicity of different drugs.

Quantitative Assessment of Ultrastructure and Light Scatter in Mouse Corneal Debridement Wounds

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Boote, Craig; Du, Yiqin; Morgan, Sian; Harris, Jonathan; Kamma-Lorger, Christina S.; Hayes, Sally; Lathrop, Kira L.; Roh, Danny S.; Burrow, Michael K.; Hiller, Jennifer; Terrill, Nicholas J.; Funderburgh, James L.; Meek, Keith M.

2012-01-01

Purpose. The mouse has become an important wound healing model with which to study corneal fibrosis, a frequent complication of refractive surgery. The aim of the current study was to quantify changes in stromal ultrastructure and light scatter that characterize fibrosis in mouse corneal debridement wounds. Methods. Epithelial debridement wounds, with and without removal of basement membrane, were produced in C57BL/6 mice. Corneal opacity was measured using optical coherence tomography, and collagen diameter and matrix order were quantified by x-ray scattering. Electron microscopy was used to visualize proteoglycans. Quantitative PCR (Q-PCR) measured mRNA transcript levels for several quiescent and fibrotic markers. Results. Epithelial debridement without basement membrane disruption produced a significant increase in matrix disorder at 8 weeks, but minimal corneal opacity. In contrast, basement membrane penetration led to increases in light scatter, matrix disorder, and collagen diameter, accompanied by the appearance of abnormally large proteoglycans in the subepithelial stroma. This group also demonstrated upregulation of several quiescent and fibrotic markers 2 to 4 weeks after wounding. Conclusions. Fibrotic corneal wound healing in mice involves extensive changes to collagen and proteoglycan ultrastructure, consistent with deposition of opaque scar tissue. Epithelial basement membrane penetration is a deciding factor determining the degree of ultrastructural changes and resulting opacity. PMID:22467580

Mini-Review: Limbal Stem Cells Deficiency in Companion Animals: Time to Give Something Back?

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Sanchez, Rick F; Daniels, Julie T

2016-04-01

Experimental animals have been used extensively in the goal of developing sight-saving therapies for humans. One example is the development of transplantation of cultured limbal epithelial stem cells (LESC) to restore vision following ocular surface injury or disease. With clinical trials of cultured LESC therapy underway in humans and a potential companion animal population suffering from similar diseases, it is perhaps time to give something back. Comparatively to humans, what is known about the healthy limbus and corneal surface physiology of companion animals is still very little. Blinding corneal diseases in animals such as symblepharon in cats with Feline Herpes Virus-1 infections require a basic understanding of the functional companion animal limbus and corneal stem cells. Our understanding of many other vision threatening conditions such as scarring of the cornea post-inflammation with lymphocytic-plasmacytic infiltrate in dogs (aka chronic superficial keratitis) or pigment proliferation with Pigmentary Keratitis of Pugs would benefit from a better understanding of the animal cornea in health and disease. This is also vital when new therapeutic approaches are considered. This review will explore the current challenges and future research directions that will be required to increase our understanding of corneal diseases in animals and consider the potential development and delivery of cultured stem cell therapy to veterinary ocular surface patients.

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

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van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Efeito do mel e do soro autólogo na cicatrização do epitélio corneano em coelhos Effect of honey and autologous serum on corneal epithelial healing in rabbits

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Gustavo Ricci Malavazzi

2005-06-01

Full Text Available OBJETIVO: Avaliar a eficácia de substâncias consideradas estimulantes da cicatrização, como o mel puro e o soro autólogo a 20% na cicatrização do epitélio corneal de coelhos. MÉTODOS: Foi realizada a remoção do epitélio corneal de dois grupos de coelhos que receberam a instilação de solução de mel puro (G1 ou soro autólogo (G2 a cada 4 horas. O olho contralateral foi usado como controle e submetido ao mesmo procedimento de remoção do epitélio, recebendo a instilação de BSS®. A área de desepitelização corneal foi avaliada 12, 24 e 48 horas após a indução do defeito epitelial. RESULTADOS: Os grupos estudados foram estatisticamente semelhantes: mel (48 horas e controle (48 horas pPURPOSE: To evaluate the efficacy of pure honey and 20% autologous serum and BSS® in corneal epithelial healing in rabbits after 48 hours. METHODS: All solutions were applied after an epithelial removal of 13-millimeters diameter area. Areas of epithelial healing were studied at 12, 24 and 48 hours. The eyes were treated every four hours during 2 days. All treated eyes were assigned to a control group (contralateral eye treated with a balanced saline solution. RESULTS: All studied groups were not significantly differents. In group one, the eyes treated with honey and the control were similar (p<0.87. In the second group the eyes treated with autologos serum and the control presented no difference in the mean score (p<0.072. CONCLUSION: Corneal epithelial healing in rabbits did not show improvement after application of either honey or autologous serum. It was possible to stabilish that the autologous serum treated eyes were clinicaly better than the control group but without statistical significance.

Vps35-deficiency impairs SLC4A11 trafficking and promotes corneal dystrophy.

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Wei Liu

Full Text Available Vps35 (vacuolar protein sorting 35 is a major component of retromer that selectively promotes endosome-to-Golgi retrieval of transmembrane proteins. Dysfunction of retromer is a risk factor for the pathogenesis of Parkinson's disease (PD and Alzheimer's disease (AD. However, Vps35/retromer's function in the eye or the contribution of Vps35-deficiency to eye degenerative disorders remains to be explored. Here we provide evidence for a critical role of Vps35 in mouse corneal dystrophy. Vps35 is expressed in mouse and human cornea. Mouse cornea from Vps35 heterozygotes (Vps35+/- show features of dystrophy, such as loss of both endothelial and epithelial cell densities, disorganizations of endothelial, stroma, and epithelial cells, excrescences in the Descemet membrane, and corneal edema. Additionally, corneal epithelial cell proliferation was reduced in Vps35-deficient mice. Intriguingly, cell surface targeting of SLC4A11, a membrane transport protein (OH- /H+ /NH3 /H2O of corneal endothelium, whose mutations have been identified in patients with corneal dystrophy, was impaired in Vps35-deficient cells and cornea. Taken together, these results suggest that SLC4A11 appears to be a Vps35/retromer cargo, and Vps35-regulation of SLC4A11 trafficking may underlie Vps35/retromer regulation of corneal dystrophy.

Implantation of Induced Pluripotent Stem Cell-Derived Tracheal Epithelial Cells.

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Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Nakamura, Ryosuke; Otsuki, Koshi; Murono, Shigeyuki; Omori, Koichi

2017-07-01

Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

Curative effect of Sodium hyaluronate and bFGF eye drops after corneal rust foreign body removal operation

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Jin-Xia Li1

2013-10-01

Full Text Available AIM: To observe the combined effect of Sodium hyaluronate eye drops and recombinant bovine basic fibroblast growth factor(bFGFeye drops on cornea epithelial repair after corneal rust foreign body extraction. METHODS: Alinety-eight cases(98 eyesof corneal rust foreign body patients were randomly distributed to combined treatment group(49 cases, 49 eyesand control group(49 cases, 49 eyes. Hyaluronate eye drops, recombinant bFGF eye drops and levofloxacin hydrochloride were applied in combined treatment group after corneal foreign body extraction. Recombinant bFGF eye drops and levofloxacin hydrochloride were applied in control group. Corneal fluorescein stain, cornea epithelial repair and local symptoms were examined thrice weekly for 2 weeks. RESULTS: General effective rate of treatment in combined group reach 96%, significantly higher than that in control group(88%, PCONCLUSION: Combined application of sodium hyaluronate eye drops and recombinant bFGF eye drops can prominently improve cornea epithelial repair after corneal lesion with proven effectiveness and safety.

Extended latanoprost release from commercial contact lenses: in vitro studies using corneal models.

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Saman Mohammadi

Full Text Available In this study, we compared, for the first time, the release of a 432 kDa prostaglandin F2a analogue drug, Latanoprost, from commercially available contact lenses using in vitro models with corneal epithelial cells. Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution (131 μg = ml solution in phosphate buffered saline. The drug release from the contact lens material and its diffusion through three in vitro models was studied. The three in vitro models consisted of a polyethylene terephthalate (PET membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC, and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment whereby, after 48 hours, between 4 to 6 μg of latanoprost (an amount well within the range of the prescribed daily dose for glaucoma patients was released. In the absence of cells, a significantly lower amount of drug, between 0.3 to 0.5 μg, was released, (p <0:001. The difference observed in release from the hydrogel lens materials in the presence and absence of cells emphasizes the importance of using an in vitro corneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes.

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

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Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Combined HLA matched limbal stem cells allograft with amniotic membrane transplantation as a prophylactic surgical procedure to prevent corneal graft rejection after penetrating keratoplasty: case report

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Paolo Capozzi

2014-09-01

Full Text Available Purpose. To determine if the use of combined HLA matched limbal stem cells allograft with amniotic membrane transplantation (AMT is a safe and effective prophylactic surgical procedure to prevent corneal graft after penetrating keratoplasty (PK. Methods. We report the case of a 17 years old patient with a history of congenital glaucoma, trabeculectomy and multiple corneal graft rejections, presenting total limbal cell deficiency. To reduce the possibility of graft rejection in the left eye after a new PK, a two step procedure was performed. At first the patient underwent a combined HLA matched limbal stem cells allograft (LAT and AMT and then, 10 months later, a new PK. Results. During 12 months of follow-up, the corneal graft remained stable and smooth, with no sign of graft rejection. Conclusions. In our patient, the prophylactic use of LAT from HLA-matched donors and AMT before PK, may result in a better prognosis of corneal graft survival.

Ocular Stem Cell Research from Basic Science to Clinical Application: A Report from Zhongshan Ophthalmic Center Ocular Stem Cell Symposium

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Hong Ouyang

2016-03-01

Full Text Available Stem cells hold promise for treating a wide variety of diseases, including degenerative disorders of the eye. The eye is an ideal organ for stem cell therapy because of its relative immunological privilege, surgical accessibility, and its being a self-contained system. The eye also has many potential target diseases amenable to stem cell-based treatment, such as corneal limbal stem cell deficiency, glaucoma, age-related macular degeneration (AMD, and retinitis pigmentosa (RP. Among them, AMD and glaucoma are the two most common diseases, affecting over 200 million people worldwide. Recent results on the clinical trial of retinal pigment epithelial (RPE cells from human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs in treating dry AMD and Stargardt’s disease in the US, Japan, England, and China have generated great excitement and hope. This marks the beginning of the ocular stem cell therapy era. The recent Zhongshan Ophthalmic Center Ocular Stem Cell Symposium discussed the potential applications of various stem cell types in stem cell-based therapies, drug discoveries and tissue engineering for treating ocular diseases.

Heterogeneity of limbal basal epithelial progenitor cells.

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Hayashida, Yasutaka; Li, Wei; Chen, Ying-Ting; He, Hua; Chen, Szu-yu; Kheirkah, Ahmad; Zhu, Ying-Tien; Matsumoto, Yukihiro; Tseng, Scheffer C G

2010-11-01

Although corneal epithelial stem cells (SCs) are located at the limbus between the cornea and the conjunctiva, not all limbal basal epithelial cells are SCs. Using 2 dispase digestions to remove different amounts of limbal basal epithelial cells for cross-sections, flat mounts, and cytospin preparations, double immunostaining to pancytokeratins (PCK) and vimentin (Vim) identified 3 p63+ epithelial progenitors such as PCK-/Vim+, PCK/Vim, and PCK-/Vim+ and 1 p63+ mesenchymal cell, PCK-/Vim+. PCK-/Vim- progenitors had the smallest cell size were 10-20 times more enriched on collagen I-coated dishes in the 5-minute rapid adherent fraction that contained the highest percentage of p63+ cells but the lowest percentage of cytokeratin12+ cells, and gave rise to high Ki67 labeling and vivid clonal growth. In contrast, PCK+/Vim+ and PCK+/Vim- progenitors were found more in the slow-adherent fraction and yielded poor clonal growth. PCK/Vim progenitors and clusters of PCK-/Vim+ mesenchymal cells, which were neither melanocytes nor Langerhans cells, were located in the limbal basal region. Therefore, differential expression of PCK and Vim helps identify small PCK-/Vim- cells as the most likely candidate for SCs among a hierarchy of heterogeneous limbal basal progenitors, and their close association with PCK-/Vim+ presumed "niche" cells.

Aloe vera: an in vitro study of effects on corneal wound closure and collagenase activity.

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Curto, Elizabeth M; Labelle, Amber; Chandler, Heather L

2014-11-01

To evaluate the in vitro effects of an aloe vera solution on (i) the viability and wound healing response of corneal cells and (ii) the ability to alter collagenase and gelatinase activities. Primary cultures of corneal epithelial cells and fibroblasts were prepared from grossly normal enucleated canine globes and treated with an aloe solution (doses ranging from 0.0-2 mg/mL). Cellular viability was evaluated using a colorimetric assay. A corneal wound healing model was used to quantify cellular ingrowth across a defect made on the confluent surface. Anticollagenase and antigelatinase activities were evaluated by incubating a bacterial collagenase/gelatinase with aloe solution (doses ranging from 0.0-500 μg/mL) and comparing outcome measures to a general metalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum (doses ranging from 0.0-100%). None of the concentrations of aloe solution tested significantly affected the viability of corneal epithelial cells or fibroblasts. Concentrations ≤175 μg/mL slightly accelerated corneal epithelial cell wound closure; this change was not significant. Concentrations ≥175 μg/mL significantly (P ≤ 0.001) slowed the rate of corneal fibroblast wound closure, while aloe concentrations Aloe solution did not alter the ability for collagenase to degrade gelatin or collagen Type I but increased the ability for collagenase to degrade Type IV collagen. Although additional experiments are required, lower concentrations of aloe solution may be beneficial in healing of superficial corneal wounds to help decrease fibrosis and speed epithelialization. An increase in collagenase activity with aloe vera warrants further testing before considering in vivo studies. © 2014 American College of Veterinary Ophthalmologists.

Stem cell factor expression after renal ischemia promotes tubular epithelial survival.

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Geurt Stokman

Full Text Available BACKGROUND: Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration of renal tissue. The cytokine stem cell factor (SCF has been shown to protect the tubular epithelium against apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse model for renal ischemia/reperfusion injury, we studied how expression of c-KIT on tubular epithelium and its ligand SCF protect cells against apoptosis. Administration of SCF specific antisense oligonucleotides significantly decreased specific staining of SCF following ischemia. Reduced SCF expression resulted in impaired renal function, increased tubular damage and increased tubular epithelial apoptosis, independent of inflammation. In an in vitro hypoxia model, stimulation of tubular epithelial cells with SCF activated survival signaling and decreased apoptosis. CONCLUSIONS/SIGNIFICANCE: Our data indicate an important role for c-KIT and SCF in mediating tubular epithelial cell survival via an autocrine pathway.

Advanced three-dimensional culture of equine intestinal epithelial stem cells.

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Stewart, A Stieler; Freund, J M; Gonzalez, L M

2018-03-01

Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Descriptive study. Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease

Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

Directory of Open Access Journals (Sweden)

Liara M Gonzalez

Full Text Available Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA, Minichromosome Maintenance Complex 2 (MCM2, Bromodeoxyuridine (BrdU and phosphorylated Histone H3 (pH3 distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2; enteroendocrine cells by Chromogranin A (CGA, Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII and sucrase isomaltase (SIM. Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.

Expression of S100B during the innate immune of corneal epithelium against fungi invasion

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Jie Zhang

2016-02-01

Full Text Available AIM: To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS: Immortalized human corneal epithelial cells (HCECs were exposed to inactive Aspergillus fumigatus (A. fumigatus conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR. S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC. RESULTS: Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn’t express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05 and continue to rise as time prolonged (P<0.01. In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05 and reached to a peak at 24h (P<0.001. Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION: S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.

Expression of S100B during the innate immune of corneal epithelium against fungi invasion

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Zhang, Jie; Zhao, Gui-Qiu; Qu, Jing; Che, Cheng-Ye; Lin, Jing; Jiang, Nan; Zhao, Han; Wang, Xue-Jun

2016-01-01

AIM To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS Immortalized human corneal epithelial cells (HCECs) were exposed to inactive Aspergillus fumigatus (A. fumigatus) conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC). RESULTS Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn't express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05) and continue to rise as time prolonged (P<0.01). In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05) and reached to a peak at 24h (P<0.001). Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection. PMID:26949634

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

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Pan, Hong; Wu, Xinyi

2012-01-01

Highlights: ► Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-β. ► Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. ► Hypoxia inhibits Acanthamoeba-induced the activation of NF-κB and ERK1/2 in HCECs. ► Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. ► LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion

Effect of the Regenerative Agent Poly(Carboxymethylglucose Sulfate) on Corneal Wound Healing After Corneal Cross-Linking for Keratoconus.

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Kymionis, George D; Liakopoulos, Dimitrios A; Grentzelos, Michael A; Tsoulnaras, Konstantinos I; Detorakis, Efstathios T; Cochener, Béatrice; Tsilimbaris, Miltiadis K

2015-08-01

To evaluate the effect of a regenerative agent (RGTA) [Cacicol20-poly(carboxymethyl glucose sulfate); OTR3, Paris, France] on corneal reepithelialization and pain after corneal cross-linking (CXL) for keratoconus. In this prospective comparative (contralateral) clinical study, patients with bilateral progressive keratoconus underwent CXL treatment. The corneal epithelium during CXL was removed using transepithelial phototherapeutic keratectomy (Cretan protocol). One eye of each patient was randomly instilled with an RGTA (Cacicol20) once a day (study group), whereas the fellow eye was instilled with artificial tears (control group). Patients were examined daily until complete reepithelialization. Postoperative examinations included slit-lamp biomicroscopy to assess the epithelial defect size and subjective evaluation of pain. The study enrolled 18 patients (36 eyes). The mean epithelial defect size for study and control groups was 19.6 ± 4.2 mm versus 21.5 ± 2.8 mm, respectively, at day 1 (P = 0.019) and 6.4 ± 3.4 mm versus 7.9 ± 4.3 mm, respectively, at day 2 (P = 0.014). At day 3 postoperatively, 61.1% of study eyes were fully reepithelialized, compared with 11.1% of control eyes (P = 0.002). RGTA (Cacicol20) instillation seems to result in faster corneal reepithelialization after CXL in this study. However, there was no significant effect in subjective pain/discomfort.

Fasudil hydrochloride, a potent ROCK inhibitor, inhibits corneal neovascularization after alkali burns in mice

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Zeng, Peng; Pi, Rong-biao; Li, Peng; Chen, Rong-xin; Lin, Li-mian; He, Hong

2015-01-01

Purpose To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice. Methods To observe the effect of fasudil, mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury, CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14. Results The incidence of CNV was significantly lower after treatment with 100 μM and 300 μM fasudil than with PBS, especially with 100 μM fasudil. Meanwhile, the incidences of corneal epithelial defects was lower (n=15, all palkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration, reduction of ROS, and upregulation of HO-1 protein after fasudil treatment. PMID:26120273

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

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Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

2014-05-29

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

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Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

2007-01-01

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

Limbal stem cell transplantation: current perspectives

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Atallah MR

2016-04-01

Full Text Available Marwan Raymond Atallah, Sotiria Palioura, Victor L Perez, Guillermo Amescua Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA Abstract: Regeneration of the corneal surface after an epithelial insult involves division, migration, and maturation of a specialized group of stem cells located in the limbus. Several insults, both intrinsic and extrinsic, can precipitate destruction of the delicate microenvironment of these cells, resulting in limbal stem cell deficiency (LSCD. In such cases, reepithelialization fails and conjunctival epithelium extends across the limbus, leading to vascularization, persistent epithelial defects, and chronic inflammation. In partial LSCD, conjunctival epitheliectomy, coupled with amniotic membrane transplantation, could be sufficient to restore a healthy surface. In more severe cases and in total LSCD, stem cell transplantation is currently the best curative option. Before any attempts are considered to perform a limbal stem cell transplantation procedure, the ocular surface must be optimized by controlling causative factors and comorbid conditions. These factors include adequate eyelid function or exposure, control of the ocular surface inflammatory status, and a well-lubricated ocular surface. In cases of unilateral LSCD, stem cells can be obtained from the contralateral eye. Newer techniques aim at expanding cells in vitro or in vivo in order to decrease the need for large limbal resection that may jeopardize the “healthy” eye. Patients with bilateral disease can be treated using allogeneic tissue in combination with systemic immunosuppressive therapy. Another emerging option for this subset of patients is the use of noncorneal cells such as mucosal grafts. Finally, the use of keratoprosthesis is reserved for patients who are not candidates for any of the aforementioned options, wherein the choice of the type of keratoprosthesis depends on

Ocular Surface Reconstruction: Recent Innovations

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Madhavan HN

2009-01-01

Full Text Available The ocular surface is exceptionally rich in complexity and functionality. Severe ocular surface disorders/conditions, such as chemical or thermal injuries, Stevens-Johnson syndrome (SJS, ocular cicatricial pemphigoid, neurotrophic keratopathy, chronic limbitis, and severe microbial keratitis cause significant morbidities and even corneal blindness. Hypofunction may be caused by Aniridia, Neurotrophy, Endocrine, Pterygium and Chronic limbitis Approximately 6000 patients are seen in Ocular Surface Clinic every year; almost 80% have some form of dry eyes. About 125 new patients of Stevens Johnson Syndrome are seen in a year of which approximately 25% may benefit from Cultured Epithelial Transplant and 75 new patients of thermal/chemical injury in a year of which almost 80% will benefit from Stem Cell Transplantation. Of the 128 severe vernal keratoconjunctivitis which were seen in the ocular surface clinic, 10% require stem cell transplantation. Nearly 30 new cases of Ocular cicatrical pemphigoid every year are seen and they may need stem cell transplantation. In addition, several patients with persistent epithelial defects may benefit from limbal stem cell transplantation to alleviate, maintain conjunctivalization regression and corneal avascularity limbal deficiency, and restore vision. Even if granted that this statistics is for a single large ophthalmic hospital, for a large country as India with 1.1 billion populations, the number of patients requiring corneal stem cell transplantation is enormous. Stem cells in the palisades of Vogt participate in regeneration and preservation of corneal transparency and avascularity. The diminished regenerative capacity seen in LSCD is characterized by persistent epithelial defects, erosion and ulceration, conjunctivalization and neovascularization, and chronic inflammation. Standard corneal transplantation for restoration of corneal clarity and avascularity is a contraindication in the surgical management

Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

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Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

2009-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/β-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

Quantitative assessment of rat corneal thickness and morphology during stem cell therapy by high-speed optical coherence tomography

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Lal, Cerine; McGrath, James; Subhash, Hrebesh; Rani, Sweta; Ritter, Thomas; Leahy, Martin

2016-03-01

Optical Coherence Tomography (OCT) is a non-invasive 3 dimensional optical imaging modality that enables high resolution cross sectional imaging in biological tissues and materials. Its high axial and lateral resolution combined with high sensitivity, imaging depth and wide field of view makes it suitable for wide variety of high resolution medical imaging applications at clinically relevant speed. With the advent of swept source lasers, the imaging speed of OCT has increased considerably in recent years. OCT has been used in ophthalmology to study dynamic changes occurring in the cornea and iris, thereby providing physiological and pathological changes that occur within the anterior segment structures such as in glaucoma, during refractive surgery, lamellar keratoplasty and corneal diseases. In this study, we assess the changes in corneal thickness in the anterior segment of the eye during wound healing process in a rat corneal burn model following stem cell therapy using high speed swept source OCT.

Activation of cytokines and NF-kappa B in corneal epithelial cells infected by respiratory syncytial virus: potential relevance in ocular inflammation and respiratory infection

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Oakes John E

2004-07-01

Full Text Available Abstract Background Respiratory syncytial virus (RSV is a major cause of lower respiratory tract infection, claiming millions of lives annually. The virus infects various cells of the respiratory tract as well as resident inflammatory cells such as macrophages. Infection activates a variety of cellular factors such as cytokines and the pro-inflammatory transcription factor, NF-kappa B, all of which are important players in the respiratory disease. However, the exact natural route of RSV infection and its etiology remain relatively unknown. In this paper, we test the hypothesis that human corneal epithelial cells, which constitute the outermost layer of the cornea, can be infected with RSV, and that the infection leads to the activation of proinflammatory macromolecules. Results Corneal swabs obtained from pediatric patients with acute respiratory disease were found to contain RSV at a high frequency (43 positive out of 72 samples, i.e., 60%. Primary corneal epithelial cells in tissue culture supported robust infection and productive growth of RSV. Infection resulted in the activation of TNF-α, IL-6 and sixteen chemokines as well as NF-κB. Three proinflammatory CXC chemokines (MIG, I-TAC, IP-10 underwent the greatest activation. Conclusions The ocular epithelium is readily infected by RSV. The pro-inflammatory cytokines are likely to play critical roles in the etiology of inflammation and conjunctivitis commonly seen in pediatric patients with respiratory infections. RSV-eye interactions have important implications in RSV transmission, immunopathology of RSV disease, and in the management of conjunctivitis.

Delayed Epithelial Closure After PRK Associated With Topical Besifloxacin Use.

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Talamo, Jonathan H; Hatch, Kathryn M; Woodcock, Emily C

2013-10-01

To report the observation of prolonged reepithelialization after photorefractive keratectomy (PRK) associated with the use of besifloxacin 0.6% (Besivance; Bausch & Lomb, Rochester, NY) underneath bandage contact lenses (BCLs) placed during surgery. An office-based private practice and retrospective chart review. The healing parameters examined included epithelial healing time, haze formation, discomfort, and visual recovery of 4 patients (7 eyes) treated with besifloxacin 0.6% under BCLs placed after the PRK was performed. All the eyes had delayed epithelial closure (mean, 8.8 days; range 5-13 days). All the patients experienced a delayed visual recovery and significant pain after the surgery, and 2 of 4 patients experienced recurrent corneal erosions for weeks to months after they underwent the PRK. All but 1 eye developed corneal haze persisting for 1 year or more after the surgery. Only 1 eye among the 7 eyes treated with besifloxacin 0.6% under the BCL had 20/20 or better uncorrected visual acuity 3 months postoperatively. All the patients treated with besifloxacin 0.6% on the stromal bed exhibited significant problems with corneal epithelial healing and delayed visual recovery. We caution the use of besifloxacin 0.6% underneath a BCL during a PRK or other ocular surface surgeries requiring corneal epithelial debridement.

Changes in corneal epithelial layer inflammatory cells in aqueous tear-deficient dry eye.

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Lin, Hui; Li, Wei; Dong, Nuo; Chen, Wensheng; Liu, Jing; Chen, Lelei; Yuan, Hongxia; Geng, Zhixin; Liu, Zuguo

2010-01-01

To investigate the morphology, distribution, and density of inflammatory cells in the corneal epithelium of aqueous tear-deficient dry eye. Thirty-two patients with non-Sjögren's syndrome (NSS) dry eye, 14 patients with Sjögren's syndrome (SS) dry eye, and 33 healthy volunteers were studied. In vivo laser scanning confocal microscopy was used to investigate both Langerhans cell (LCs) and leukocyte distribution and density in the peripheral and central corneal epithelium. LC morphology was also evaluated. Multifactor regression analysis assessed whether there is a correlation between clinical manifestations and inflammatory cell densities. LCs were present in both central (34.9 +/- 5.7 cells/mm(2)) and peripheral (90.7 +/- 8.2 cells/mm(2)) parts of the normal corneal epithelium. Moreover, LC density increased dramatically in the central corneal epithelium in patients with NSS (89.8 +/- 10.8 cells/mm(2)) and SS (127.9 +/- 23.7 cells/mm(2)). The ratio of LCs with obvious processes was much higher in patients with dry eye than in healthy volunteers. LC density also increased in peripheral corneal epithelium in patients with SS, but not in those with NSS. Leukocyte density in normal corneal epithelium was very low, whereas it increased in the central corneal epithelium (4.6 +/- 1.0 cells/mm(2)) in NSS and in both central (49.0 +/- 12.9 cells/mm(2)) and peripheral (84.2 +/- 36.8 cells/mm(2)) corneal epithelium in SS. Densities of LCs and leukocytes showed significant correlation with the severity found in clinical evaluation. The LC and leukocyte changes in the corneal epithelium suggest their involvement in aqueous tear-deficient dry eye pathophysiology. In vivo dynamic assessment of central corneal inflammatory cell density may serve as an indicator of dry eye severity and provide new insight for dry eye treatment.

SUITABILITY OF CORNEAL TISSUE FOR TRANSPLANTATION PROCURED FROM HANGING CASES

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Rekha Gyanchand

2017-06-01

Full Text Available BACKGROUND Requirement of donor cornea is essential to target the corneal blind. The best method to procure such corneas is from any major hospitals, which has a mortuary facility. The eye donation with hanging as the cause of death is very common in a mortuary setup. Some factors that are concerning regarding corneas procured from death due to hanging is the prolonged exposure of the cornea at the time of death, the exact time of death is not known, most of the cadavers are refrigerated for investigations as these arrive at the mortuary usually at night. Due to these reasons, the corneal surgeons are hesitant to use corneas procured from death due to hanging for corneal transplantation. Analysing these corneas would contribute to a great extent to the donor cornea pool in providing sight to the corneal blind, especially as majority are young individuals who commit suicide by hanging. In this study, the donor corneas were analysed with regards to corneal epithelial defect, endothelial cell morphology and utilisation of these corneas for transplantation. The aim of the HCRP study is to analyse the effect of death due to hanging on donor cornea. 1. Corneal epithelial status. 2. Corneal endothelial cell morphology. 3. Utilisation of corneas for transplantation. MATERIALS AND METHODS Donor corneas from 22 donors who died due to hanging were procured from hospital mortuary. All the 44 corneas were transplanted. Various parameters like demography, death to enucleation time, cadaver preservation in cold storage, endothelial cell density and utilisation of cornea for transplantation were noted. Design- Retrospective study. Statistical Analysis- Descriptive statistics, Pearson and Spearman correlation and Chi-square test were used to test the hypotheses. RESULTS Out of the 44 corneas analysed, 75% of the donors were refrigerated as a part of medicolegal investigations protocol. The average DTP time was 12 hours in refrigerated group and 5 hours in non

Corneal thickness changes during corneal collagen cross-linking with UV-A irradiation and hypo-osmolar riboflavin in thin corneas

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Belquiz Amaral Nassaralla

2013-06-01

Full Text Available PURPOSE: To evaluate the thinnest corneal thickness changes during and after corneal collagen cross-linking treatment with ultraviolet-A irradiation, using hypo-osmolar riboflavin solution in thin corneas. METHODS: Eighteen eyes of 18 patients were included in this study. After epithelium removal, iso-osmolar 0.1% riboflavin solution was instilled to the cornea every 3 minutes for 30 minutes. Hypo-osmolar 0.1% riboflavin solution was then applied every 20 seconds for 5 minutes or until the thinnest corneal thickness reached 400 µm. Ultraviolet-A irradiation was performed for 30 minutes. During irradiation, iso-osmolar 0.1% riboflavin drops were applied every 5 minutes. Ultrasound pachymetry was performed at approximately the thinnest point of the cornea preoperatively, after epithelial removal, after iso-osmolar riboflavin instillation, after hypo-osmolar riboflavin instillation, after ultraviolet-A irradiation, and at 1, 6 and 12 months after treatment. RESULTS: Mean preoperative thinnest corneal thickness was 380 ± 11 µm. After epithelial removal it decreased to 341 ± 11 µm, and after 30 minutes of iso-osmolar 0.1% riboflavin drops, to 330 ± 7.6 µm. After hypo-osmolar 0.1% riboflavin drops, mean thinnest corneal thickness increased to 418 ± 11 µm. After UVA irradiation, it was 384 ± 10 µm. At 1, 6 and 12 months after treatment, it was 372 ± 10 µm, 381 ± 12.7, and 379 ± 15 µm, respectively. No intraoperative, early postoperative, or late postoperative complications were noted. CONCLUSIONS: Hypo-osmolar 0.1% riboflavin solution seems to be effective for swelling thin corneas. The swelling effect is transient and short acting. Corneal thickness should be monitored throughout the procedure. Larger sample sizes and longer follow-up are required in order to make meaningful conclusions regarding safety.

Caveolin-1 as a Novel Indicator of Wound-Healing Capacity in Aged Human Corneal Epithelium

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Rhim, Ji Heon; Kim, Jae Hoon; Yeo, Eui-Ju; Kim, Jae Chan; Park, Sang Chul

2010-01-01

Excess caveolin-1 has been reported to play a role in age-dependent hyporesponsiveness to growth factors in vitro. Therefore, we hypothesized that caveolin-1–dependent hyporesponsiveness to growth factors in aged corneal epithelial cells might be responsible for delayed wound healing in vivo. To test this hypothesis, we evaluated corneal wound-healing time by vital staining using fluorescein after laser epithelial keratomileusis (LASEK). We compared wound-healing times in young, middle-aged a...

Epithelial WNT Ligands Are Essential Drivers of Intestinal Stem Cell Activation

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Winnie Y. Zou

2018-01-01

Full Text Available Intestinal stem cells (ISCs maintain and repair the intestinal epithelium. While regeneration after ISC-targeted damage is increasingly understood, injury-repair mechanisms that direct regeneration following injuries to differentiated cells remain uncharacterized. The enteric pathogen, rotavirus, infects and damages differentiated cells while sparing all ISC populations, thus allowing the unique examination of the response of intact ISC compartments during injury-repair. Upon rotavirus infection in mice, ISC compartments robustly expand and proliferating cells rapidly migrate. Infection results specifically in stimulation of the active crypt-based columnar ISCs, but not alternative reserve ISC populations, as is observed after ISC-targeted damage. Conditional ablation of epithelial WNT secretion diminishes crypt expansion and ISC activation, demonstrating a previously unknown function of epithelial-secreted WNT during injury-repair. These findings indicate a hierarchical preference of crypt-based columnar cells (CBCs over other potential ISC populations during epithelial restitution and the importance of epithelial-derived signals in regulating ISC behavior.

Acceleration of vertical migration of corneal epithelial cells in albino rats during chronic immobilization stress

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Timoshin, S.S.; Berezhnova, N.I.

1986-01-01

This paper studies the effect of chronic immobilization stress on the kinetics of corneal epithelial cells from the basal layer into higher layers. Experiments were carried out on 49 male rats. The animals were given an intraperitoneal injection of tritium-thymidine and an additional application of 5 microCi of tritium-thymidine was made to its surface because the cornea has no blood supply. The animals were killed and the cornea removed for investigation. Values of the index of labeled nuclei and intensity of thymidine labeling, characterizing DNA synthesis in the corneas of the control and experimental animals showed no significant change compared with their values in a pervious series of experiments. Chronic exposure to stress increased the velocity of vertical migration of the cells from the basal layer toward the outer layers of the cornea

Transplante de células-tronco epiteliais límbicas alógenas expandidas ex vivo sobre membrana amniótica: relato de caso Transplantation of allogenic limbal epithelial stem cells cultivated ex vivo on amniotic membrane: case report

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José Álvaro Pereira Gomes

2009-04-01

Full Text Available Paciente apresentou falência de transplante de limbo e conjuntiva de doador vivo alógeno no olho direito após ceratoconjuntivite epidêmica. Após alguns meses, foi submetida a transplante de células-tronco epiteliais límbicas alógenas cultivadas ex vivo sobre membrana amniótica (primeiro caso no Brasil, tendo evoluído com epitelização total da córnea e melhora da acuidade visual. Após o 3º mês da cirurgia, iniciou-se neovascularização superficial periférica com piora da transparência corneana. A visão manteve-se 0,1 após um ano de cirurgia.Case report of a patient who developed failure of an allogenic living related conjunctival limbal transplantation in the right eye after an episode of epidemic keratoconjunctivitis. After a few months, she underwent transplantation of allogenic limbal epithelial stem cells cultivated ex vivo on amniotic membrane (first case in Brazil. The patient evolved with total corneal epithelialization and improvement of the visual acuity. Three months after the surgery, peripheral superficial neovascularization with worsening of the corneal transparency was observed. The vision remained 0.1 after one year of the transplantation.

Induction of corneal collagen cross-linking in experimental corneal alkali burns in rabbits

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Marcello Colombo-Barboza

2014-10-01

Full Text Available Objective: To evaluate the effect of riboflavin-ultraviolet-A-induced cross-linking (CXL following corneal alkali burns in rabbits. Methods: The right corneas and limbi of ten rabbits were burned using a 1N solution of NaOH and the animals were then divided into two groups: a control group submitted to clinical treatment alone and an experimental group that was treated 1 h after injury with CXL, followed by the same clinical treatment as administered to the controls. Clinical parameters were evaluated post-injury at 1, 7, 15, and 30 days by two independent observers. Following this evaluation, the corneas were excised and examined histologically. Results: There were no statistically significant differences in clinical parameters, such as hyperemia, corneal edema, ciliary injection, limbal ischemia, secretion, corneal neovascularization, symblepharon, or blepharospasm, at any of the time-points evaluated. However, the size of the epithelial defect was significantly smaller in the CXL group (p<0.05 (day 15: p=0.008 and day 30: p=0.008 and the extent of the corneal injury (opacity lesion was also smaller (day 30: p=0.021. Histopathology showed the presence of collagen bridges linking the collagen fibers in only the CXL group. Conclusions: These results suggest that the use of CXL may improve the prognosis of acute corneal alkali burns.

Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health.

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Xiang, Ping; Liu, Rong-Yan; Li, Chao; Gao, Peng; Cui, Xin-Yi; Ma, Lena Q

2017-11-01

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC 50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas. Published by Elsevier Ltd.

Explore the full thick layer of corneal transplantation in the treatment of pseudomonas aeruginosa corneal ulcer infection

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Xin Wang

2015-02-01

Full Text Available AIM: To explore the feasibility, safety and effect of the full-thickness lamellar keratoplasty for the treatment of pseudomonas aeruginosa corneal ulcer. METHODS: Based on a retrospective non-controlled study, 25 patients were given the full-thickness lamellar keratoplasty for clinical diagnosis of pseudomonas aeruginosa infection and corneal ulcer medication conventional anti-gram-negative bacteria. Routine follow-up were carried out at postoperative 1wk; 1, 3, 6, 12, 18mo to observe the situation of corneal epithelial healing, recurrent infection, immune rejection, graft transparency and best corrected visual acuity, etc. At the 6 and 12mo postoperative, corneal endothelial cell density was reexamined.RESULTS: No patients because of Descemet's membrane rupture underwent penetrating keratoplasty surgery: One only in cases of bacterial infection after 1mo, once again did not cultivate a culture of bacteria pseudomonas aeruginosa, and the remaining 24 cases average follow-up 14±6mo, corneal graft were transparent, the cure rate was 96%. At the sixth month after surgery, there were 16 cases of eye surgery best corrected visual acuity ≥4.5, of which 3 cases ≥4.8. At the sixth month after surgery, the average corneal endothelial cell density 2 425±278/mm2; At 12mo postoperatively, it was 2 257± 326/mm2.CONCLUSION: Full-thickness lamellar keratoplasty is an effective method of pseudomonas aeruginosa infection in the treatment of corneal ulcers, corneal drying material glycerol can be achieved by visual effects.

Serum from plasma rich in growth factors regenerates rabbit corneas by promoting cell proliferation, migration, differentiation, adhesion and limbal stemness.

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Etxebarria, Jaime; Sanz-Lázaro, Sara; Hernáez-Moya, Raquel; Freire, Vanesa; Durán, Juan A; Morales, María-Celia; Andollo, Noelia

2017-12-01

To evaluate the regenerating potential and the mechanisms through which the autologous serum derived from plasma rich in growth factors (s-PRGF) favours corneal wound healing in vitro and in vivo. We compared the effect of various concentrations of s-PRGF versus fetal bovine serum (FBS) and control treatment in rabbit primary corneal epithelial and stromal cells and wounded rabbit corneas. Cell proliferation was measured using an enzymatic colorimetric assay. In vitro and in vivo wound-healing progression was assessed by image-analysis software. Migration and invasion were evaluated using transfilter assays. Histological structure was analysed in stained sections. Protein expression was evaluated by immunohistochemistry. s-PRGF promoted the robust proliferation of epithelial cultures at any concentration, similar to FBS. Likewise, s-PRGF and FBS produced similar re-epithelialization rates in in vitro wound-healing assays. In vivo, s-PRGF treatment accelerated corneal wound healing in comparison with control treatment. This difference was significant only for 100% s-PRGF treatment in our healthy rabbit model. Histological analysis confirmed normal epithelialization in all cases. Immunohistochemistry showed a higher expression of cytokeratins 3/76 and 15, zonula occludens-1 and alpha-smooth muscle actin proteins as a function of s-PRGF concentration. Notably, keratocyte density in the anterior third of the stroma increased with increase in s-PRGF concentration, suggesting an in vivo chemotactic effect of s-PRGF on keratocytes that was further confirmed in vitro. s-PRGF promotes proliferation and migration and influences limbal stemness, adhesion and fibrosis during corneal healing. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components.

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Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun

2011-01-01

To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were measured using 3-(4,5)-dimethylthiahiazo (-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and transwell migration assay. Cell damage was determined with the lactate dehydrogenase (LDH) assay. Cell viability and median lethal time (LT₅₀) were measured by 7-amino-actinomycin D (7-AAD) staining and flow cytometry analysis. Cellular effects after exposure of HCECs to the four anti-inflammatory eye drops were concentration dependent. The differences of cellular toxicity on cell proliferation became significant at lower concentrations (Eye Drops showed significant increasing effects on cell damage and viability when compared with the other three solutions. Tobramycin & Dex Eye Drops inhibited the migration of HCECs significantly. Tobramycin & Dex Eye Drops showed the quickest effect on cell viability: the LT₅₀ was 3.28, 9.23, 10.38, and 23.80 min for Tobramycin & Dex Eye Drops, Diclofenac Sodium Eye Drops, Pranoprofen Eye Drops, and Bromfenac Sodium Hydrate Eye Drops, respectively. However, the comparisons of cellular toxicity revealed significant differences between the eye drops and their active components under the same concentration. The corneal epithelial toxicity differences among the active components of the four eye drops became significant as higher concentration (>0.020%). The four anti-inflammatory eye drops showed different cellular effects on HCECs, and the toxicity was not related with their active components, which provides new reference for the clinical application and drug

Effect of polysaccharide of dendrobium candidum on proliferation and apoptosis of human corneal epithelial cells in high glucose

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Li, Qiangxiang; Chen, Jing; Li, Yajia; Chen, Ting; Zou, Jing; Wang, Hua

2017-01-01

Abstract Background: The aim of the study was to observe the effect of polysaccharide of dendrobium candidum (PDC) and high glucose on proliferation, apoptosis of human corneal epithelial cells (HCEC). Methods: The MTT method was used to screen and take the optimal high-glucose concentration, treatment time, and PDC concentration using HCEC and divide it into 4 groups: control group (C), high glucose group (HG), PDC group, and HG + PDC group. We observed and compared the effect of the 4 groups on HCEC proliferation by MTT, apoptosis by Annexin V-FITC/PI double fluorescent staining and flow cytometry (FCM), and expression of bax mRNA and bcl-2 mRNA by RT-qPCR. Results: Compared with the control group, proliferative activity of HCEC cells was reduced; the cells apoptosis ratio was increased; the expression of bax mRNA was increased, and the expression of bcl-2 mRNA was reduced in the HG group. Proliferative activity of HCEC cells in the PDC group was increased, and the expression of bcl-2 mRNA was increased but that of bax mRNA was decreased. Proliferative activity of HCEC cells in the HG + PDC group was increased, but it could not restore to the normal level; the expression of bax mRNA was significantly decreased but the expression of bcl-2 mRNA was significantly increased. Conclusions: Our results demonstrate that high glucose can inhibit proliferative activity and induce apoptosis of HCEC. PDC can improve the proliferative activity of HCEC cells under the high glucose environment and reduce the apoptosis of cells by regulating the expression of bax and bcl-2. PDC play a very important role on protecting and repairing of corneal epithelial cells damage in high glucose. PMID:28796073

Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model.

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Khoh-Reiter, Su; Jessen, Bart A

2009-07-28

Benzalkonium chloride (BAC) is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D) corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC) and olopatadine (0.01% BAC) was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T) cell cultures, expression levels (mRNA and protein) of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 microL drops twice daily in 1 eye for 1 year) in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC) and untreated eyes. The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC contained in ophthalmic solutions are not likely to cause

Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model

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Jessen Bart A

2009-07-01

Full Text Available Abstract Background Benzalkonium chloride (BAC is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. Methods The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC and olopatadine (0.01% BAC was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T cell cultures, expression levels (mRNA and protein of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 μL drops twice daily in 1 eye for 1 year in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. Results In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC and untreated eyes. Conclusion The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC

A study comparing standard and transepithelial collagen cross-linking riboflavin solutions: epithelial findings and pain scores.

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Yuksel, Erdem; Novruzlu, Shahin; Ozmen, Mehmet C; Bilgihan, Kamil

2015-06-01

To evaluate epithelial signs and pain after epithelial-on corneal collagen cross-linking (Epi-on CCL) with new transepithelial riboflavin formulation and epithelial-off corneal collagen cross-linking (Epi-off CCL) with standard riboflavin formulation and to compare pain and duration of epithelial healing between both techniques. Thirty-nine eyes of 39 patients undergoing Epi-on CCL and 39 eyes of 39 patients undergoing Epi-off CCL were evaluated. Corneal epithelial signs and durations of corneal epithelial healing and subjective pain scores after the procedures were recorded and compared between 2 groups. Total epithelialization was observed after 2.7 ± 0.7 days in Epi-on CCL and 2.3 ± 0.4 days in Epi-off CCL (P = 0.006). The mean pain score on the first day was 3.1 ± 0.6 in Epi-on CCL and 2.3 ± 0.4 in Epi-off CCL with a significant difference (P = 0.0001). The epithelial damage was observed in both procedures; also, the epithelial healing time was longer in Epi-on CCL and it is of great importance that the patients should have therapeutic contact lenses until the epithelium heals in both procedures. The Epi-off CCL group had less pain scores than the Epi-on CCL group and more pain problems after Epi-on CCL still remains. The patient should be informed about pain, even if the Epi-on CCL procedure was performed.

Toxic corneal epitheliopathy after intravitreal methotrexate and its treatment with oral folic acid.

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Gorovoy, Ian; Prechanond, Tidarat; Abia, Maravillas; Afshar, Armin R; Stewart, Jay M

2013-08-01

To determine whether oral folic acid can ameliorate an iatrogenic, visually significant corneal epitheliopathy, which commonly occurs with intravitreal injections of methotrexate for the treatment of intraocular lymphoma. We report 2 cases of visually significant corneal epitheliopathy occurring after intravitreal injections of methotrexate for intraocular lymphoma. The first patient did not receive any treatment for the corneal disease, and the second patient with bilateral intraocular lymphoma received 1 mg of oral folic acid daily, a commonly used dosage for patients on systemic methotrexate. In the first patient without treatment, there was a complete regression of the corneal epithelial disease only when the frequency of intravitreal methotrexate was reduced from weekly to monthly as per a commonly used dosage regimen for methotrexate. In the second patient, the corneal disease improved 80% within 1 week of initiating oral folic acid for her eye already experiencing severe epitheliopathy during her weekly dosing regimen of methotrexate and also had significantly decreased epithelial disease in her second eye that started weekly intravitreal methotrexate several weeks after beginning oral folic acid. Currently, oral folic acid supplements are recommended for patients using systemic methotrexate to minimize drug toxicity. We suggest a similar use in patients undergoing intravitreal methotrexate injections to decrease toxic effects on the corneal epithelium.

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals.

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Takezawa, Toshiaki; Nishikawa, Kazunori; Wang, Pi-Chao

2011-09-01

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman's membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test. Copyright © 2011 Elsevier Ltd. All rights reserved.

A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny

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Marcela Aldrovani

Full Text Available ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

Differentiation of Pluripotent Stem Cells to Retinal Pigment Epithelial Cells: An Approach Toward Retinal Degenerative Diseases Treatment

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Maryam Parvini

2013-10-01

Full Text Available Pluripotent stem cells as the cells with a capacity for self-renewal and differentiation into various specificcell types have been highly regarded in regenerative medicine studies. To repair the eye disease damages, thedifferentiation into retinal pigment epithelial cells of pluripotent stem cells has gained great importance inrecent decades because the inappropriate function of these cells is the main cause of degenerative diseases suchas the age-related macular degeneration. Millions of people in the world suffer this disease.To restore the damaged cells and, finally, to improve the vision, numerous studies have been conducted on usingpluripotent stem cells, their differentiation into retinal pigment epithelial cells, and finally, their applicationin cell therapy. Based on this, many researchers have attempted to produce highly efficient retinal pigmentepithelial cells, such that they show a proper function after transplant, along with the host cells. In this reviewarticle, the importance and the role of pigment epithelial cells, as well as, the studies on the in vitro productionof these cells were examined

Quantitative analysis of corneal stromal riboflavin concentration without epithelial removal.

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Rubinfeld, Roy S; Stulting, R Doyle; Gum, Glenwood G; Talamo, Jonathan H

2018-02-01

To compare the corneal stromal riboflavin concentration and distribution using 2 transepithelial corneal crosslinking (CXL) systems. Absorption Systems, San Diego, California, USA. Experimental study. The stromal riboflavin concentration of 2 transepithelial CXL systems was compared in rabbit eyes in vivo. The systems were the Paracel/Vibex Xtra, comprising riboflavin 0.25% solution containing TRIS and ethylenediaminetetraacetic acid and an isotonic solution of riboflavin 0.25%, (Group 1) and the CXLO system (Group 2). Manufacturers' Instructions For Use were followed. The intensity of riboflavin fluorescence by slitlamp observation 10, 15, and 20 minutes after instillation was graded on a scale of 0 to 5. The animals were humanely killed and the corneal stromal samples analyzed with liquid chromatography and mass spectrometry. The mean riboflavin fluorescence intensity grades in Group 1 (4 eyes) were 3.8, 4.8, and 4.8 at 10, 15, and 20 minutes, respectively. The mean grades in Group 2 (3 eyes) were 2.0, 2.3, and 2.0, respectively. The riboflavin distribution was uniform in Group 1 but not in Group 2. The mean riboflavin concentration by liquid chromatography and mass spectrometry was 27.0 μg/g stromal tissue in Group 1 and 6.7 μg/g in Group 2. A stromal riboflavin concentration theoretically adequate for CXL, 15 μg/g, was achieved in all eyes in Group 1 and no eyes in Group 2. Slitlamp grading correlated well with liquid chromatography and mass spectrometry concentration (R 2  = 0.940). The system used in Group 1 produced corneal riboflavin concentrations that were theoretically adequate for effective transepithelial CXL (≥15 μg/g), while the system in Group 2 did not. Slitlamp grading successfully estimated the corneal riboflavin concentration and can be used to ensure an adequate concentration of riboflavin in the cornea for transepithelial CXL. Copyright © 2018 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Corneal epithelial cell viability of an ex vivo porcine eye model.

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Chan, Ka Yin; Cho, Pauline; Boost, Maureen

2014-07-01

The aim was to assess the consistency of corneal epithelial cell viability of an ex vivo porcine eye model. Six porcine eye models (four test and two control) were prepared for each experiment. The model has a computer-controlled mechanical arm, which could move the eyelid of the porcine eye and apply phosphate buffered saline to simulate blinking and lacrimation. The four test eyes were set up to simulate evaporative dry eyes with simulated lacrimation and blinking (one blink and one drop of buffered saline per minute) over three hours. Control A models were set up to collect pre-experimental baseline data, while those of control B were the same as the test eyes but without lacrimation and blinking simulation. All porcine eyes were kept in a closed chamber with temperature and humidity well controlled. After three hours, the cells of all eyes (except control A, which were assessed immediately before commencement of the experiment) were assessed. The eyes were first dipped into 0.4 per cent trypan blue solution. Following the dissection and separation of the cells, the number of dead cells were then counted under the microscope with a field size of 0.25 mm(2). The experiment was repeated 11 times. No significant differences were found in the number of dead cells among the four test eyes in both the central and peripheral cornea. There were significantly more dead cells in the test eyes compared to control A but significantly less when compared to control B. More dead cells were found in the central cornea than the peripheral cornea in the test eyes but the difference was not observed in controls A and B. Epithelial cell viabilities among the four porcine eye models with simulated lacrimation and blinking were consistent. The majority of cells were viable before the experiment and simulated lacrimation and blinking maintained more viable cells over time. © 2014 The Authors. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.

Indian hedgehog regulates intestinal stem cell fate through epithelial-mesenchymal interactions during development

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Kosinski, C.; Stange, D.E.; Xu, C.; Chan, A.S.; Ho, C.; Yuen, S.T.; Mifflin, R.C.; Powell, D.W.; Clevers, H.; Leung, S.Y.; Chen, X.N.

2010-01-01

BACKGROUND & AIMS: Intestinal stem cells (ISCs) are regulated by the mesenchymal environment via physical interaction and diffusible factors. We examined the role of Indian hedgehog (Ihh) in mesenchymal organization and the mechanisms by which perturbations in epithelial-mesenchymal interactions

Effects of the Loss of Conjunctival Muc16 on Corneal Epithelium and Stroma in Mice

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Shirai, Kumi; Okada, Yuka; Cheon, Dong-Joo; Miyajima, Masayasu; Behringer, Richard R.; Yamanaka, Osamu; Saika, Shizuya

2014-01-01

Purpose. To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. Methods. We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. Results. The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. Conclusions. The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome. PMID:24812549

Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates

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Mohammad Moad

2017-08-01

Full Text Available Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1 enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

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Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

2014-04-01

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

Determinants of the epithelial-muscular axis on embryonic stem cell-derived gut-like structures.

Science.gov (United States)

Luo, Yi; Takaki, Miyako; Misawa, Hiromi; Matsuyoshi, Hiroko; Sasahira, Tomonori; Chihara, Yoshitomo; Fujii, Kiyomu; Ohmori, Hitoshi; Kuniyasu, Hiroki

2010-01-01

Dome-like structures with epithelial-muscular layers resembling the gut have been derived from mouse embryonic stem (ES) cells. These domes have been reported to show spontaneous contractions and are called ES gut. In the present study, we examined the epithelial-muscular axis of these domes by detecting differentiation markers. A normal epithelial-muscular axis was exhibited in the domes with spontaneous motility, whereas the domes without spontaneous motility showed either an inverted or obscure axis. To investigate the factors affecting the epithelial-muscular axis, we examined the expression of hedgehog signaling factors in the domes. Expression of hedgehog family factors was detected in the epithelial components of the domes with motility, whereas this expression was inverted or obscure in the domes without motility. Out of the 25 domes, 10 of the 10 motility (+) domes showed a normal epithelial-muscular axis, whereas 14 of the 15 motility (-) domes lacked a normal epithelial-muscular axis. This implies that activin A upregulated the expression of sonic hedgehog and intestinal alkaline phosphatase in the embryoid bodies. These findings suggest that the motility of the ES gut depends on the domes' epithelial-muscular axis. Copyright © 2010 S. Karger AG, Basel.

[Quantitative analysis of the corneal subbasal nerves in different degrees of dry eye with AutoCAD].

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Cheng, Y; Wu, J; Zhu, H F; Cheng, Y; Zhu, X P

2016-03-01

To evaluate the practical value of AutoCAD in quantitative analysis of corneal subbasal epithelial nerves with different degrees of dry eye. Ninety patients were divided into groups of mild, moderate, and severe dry eye, 30 patients (60 eyes) in each group. And 30 healthy volunteers were recruited as the normal control group. Confocal microscopy was used to observe the length of the subbasal epithelial nerve plexus. The images were analyzed by AutoCAD software to determine the density (mm/mm(2)), the number of branches, and the curvature score of the subbasal epithelial nerves. These data of patients with dry eye and the controls were statistically compared, by analysis of variance(ANOV). By AutoCAD software, quantitative analysis of the corneal subbasal epithelial nerves was successfully performed. The nerve density in the patients with mild dry eye[(16.70±3.43) mm/mm(2)] was not significantly different from the controls[(15.87 ± 2.75) mm/mm(2)] (P=0.880), but the number of nerval branches 13.43±2.46 and the curvature 3.10±0.80 increased significantly (Pdry eye was significantly different from that in the normal control group (F=114.739, Pdry eye than the controls, but there was no significant difference in the curvature scores between the two groups (P= 0.557). AutoCAD software is useful in the quantitative analysis of corneal nerve images under a confocal microscope. The corneal subbasal epithelial nerve density, the number of branches, and the curvature of the nerves are related to the degree of dry eye, and may be used as clinical indicators.

Caveolin-1 as a novel indicator of wound-healing capacity in aged human corneal epithelium.

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Rhim, Ji Heon; Kim, Jae Hoon; Yeo, Eui-Ju; Kim, Jae Chan; Park, Sang Chul

2010-01-01

Excess caveolin-1 has been reported to play a role in age-dependent hyporesponsiveness to growth factors in vitro. Therefore, we hypothesized that caveolin-1-dependent hyporesponsiveness to growth factors in aged corneal epithelial cells might be responsible for delayed wound healing in vivo. To test this hypothesis, we evaluated corneal wound-healing time by vital staining using fluorescein after laser epithelial keratomileusis (LASEK). We compared wound-healing times in young, middle-aged and elderly patients. We also examined caveolin-1 levels and other aging markers, such as p53 and p21, in the corneal epithelium. Elderly patients generally had higher caveolin-1 levels in the corneal epithelia than young patients. There were, however, variations among individuals with increased caveolin-1 in some young patients and decreased levels in some elderly patients. Wound-healing time after LASEK correlated well with the corneal caveolin-1 status. Therefore, we suggest that caveolin-1 status might be responsible for delayed wound healing in elderly patients after LASEK. Caveolin-1 status might be a regulator for wound-healing capacity and a novel target for in vivo adjustment.

Topical timolol with and without benzalkonium chloride: epithelial permeability and autofluorescence of the cornea in glaucoma.

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de Jong, C; Stolwijk, T; Kuppens, E; de Keizer, R; van Best, J

1994-04-01

Epithelial permeability and autofluorescence of the cornea were determined by fluorophotometry in 21 patients with open-angle glaucoma or ocular hypertension using timolol medication with the preservative benzalkonium chloride (BAC) and 2 weeks after changing to timolol medication without BAC. The investigation was performed to determine whether removal of BAC would reduce toxic effects on the cornea and complaints of sensations of burning or dry eye. Corneal epithelial permeability decreased significantly after changing medication (mean decrease per patient 27%, P = 0.025). Corneal autofluorescence increased significantly after changing medication suggesting an alteration in corneal metabolism (mean increase per patient 6%, P = 0.003). Timolol without BAC was found to be as effective as timolol with BAC in reducing intraocular pressure (P = 0.4). Removal of BAC from timolol resulted in an improvement of corneal epithelial barrier function and in a reduction of complaints. The improvement was found to be proportional to the duration of the preceding BAC-containing therapy.

Comparison of Changes in Central Corneal Thickness During Corneal Collagen Cross-Linking, Using Isotonic Riboflavin Solutions With and Without Dextran, in the Treatment of Progressive Keratoconus.

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Zaheer, Naima; Khan, Wajid Ali; Khan, Shama; Khan, M Abdul Moqeet

2018-03-01

To compare intraoperative changes in central corneal thickness (CCT) during corneal cross-linking, using 2 different isotonic riboflavin solutions either with dextran or with hydroxy propyl methylcellulose, in the treatment of progressive keratoconus. In this retrospective study, we analyzed records of corneal thickness measurements, taken during various steps of cross-linking. Cross-linking was performed using either isotonic riboflavin with dextran (group A) or isotonic riboflavin with hydroxy propyl methylcellulose (without dextran) (group B). CCT measurements were recorded before and after epithelial removal, after saturation with respective isotonic riboflavin solution, after use of hypotonic riboflavin in selected cases, and after ultraviolet A (UV-A) application. A mixed-way analysis of variance was conducted on CCT readings within each group and between both groups, and p dextran causes a significant decrease in corneal thickness, whereas dextran-free isotonic riboflavin causes a significant increase in corneal thickness, thus facilitating the procedure.

The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

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Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

2017-08-01

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

Ebselen Preserves Tissue-Engineered Cell Sheets and their Stem Cells in Hypothermic Conditions.

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Katori, Ryosuke; Hayashi, Ryuhei; Kobayashi, Yuki; Kobayashi, Eiji; Nishida, Kohji

2016-12-14

Clinical trials have been performed using autologous tissue-engineered epithelial cell sheets for corneal regenerative medicine. To improve stem cell-based therapy for convenient clinical practice, new techniques are required for preserving reconstructed tissues and their stem/progenitor cells until they are ready for use. In the present study, we screened potential preservative agents and developed a novel medium for preserving the cell sheets and their stem/progenitor cells; the effects were evaluated with a luciferase-based viability assay. Nrf2 activators, specifically ebselen, could maintain high ATP levels during preservation. Ebselen also showed a strong influence on maintenance of the viability, morphology, and stem cell function of the cell sheets preserved under hypothermia by protecting them from reactive oxygen species-induced damage. Furthermore, ebselen drastically improved the preservation performance of human cornea tissues and their stem cells. Therefore, ebselen shows good potential as a useful preservation agent in regenerative medicine as well as in cornea transplantation.

Congenital Corneal Anesthesia and Neurotrophic Keratitis: Diagnosis and Management

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Flavio Mantelli

2015-01-01

Full Text Available Neurotrophic keratitis (NK is a rare degenerative disease of the cornea caused by an impairment of corneal sensory innervation, characterized by decreased or absent corneal sensitivity resulting in epithelial keratopathy, ulceration, and perforation. The aetiopathogenesis of corneal sensory innervation impairment in children recognizes the same range of causes as adults, although they are much less frequent in the pediatric population. Some extremely rare congenital diseases could be considered in the aetiopathogenesis of NK in children. Congenital corneal anesthesia is an extremely rare condition that carries considerable diagnostic and therapeutic problems. Typically the onset is up to 3 years of age and the cornea may be affected in isolation or the sensory deficit may exist as a component of a congenital syndrome, or it may be associated with systemic somatic anomalies. Accurate diagnosis and recognition of risk factors is important for lessening long-term sequelae of this condition. Treatment should include frequent topical lubrication and bandage corneal or scleral contact lenses. Surgery may be needed in refractory cases. The purpose of this review is to summarize and update data available on congenital causes and treatment of corneal hypo/anesthesia and, in turn, on congenital NK.

Lycium barbarum Polysaccharides Protect Rat Corneal Epithelial Cells against Ultraviolet B-Induced Apoptosis by Attenuating the Mitochondrial Pathway and Inhibiting JNK Phosphorylation

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Shaobo Du

2017-01-01

Full Text Available Lycium barbarum polysaccharides (LBPs have been shown to play a key role in protecting the eyes by reducing the apoptosis induced by certain types of damage. However, it is not known whether LBPs can protect damaged corneal cells from apoptosis. Moreover, no reports have focused on the role of LBPs in guarding against ultraviolet B- (UVB- induced apoptosis. The present study aimed to investigate the protective effect and underlying mechanism of LBPs against UVB-induced apoptosis in rat corneal epithelial (RCE cells. The results showed that LBPs significantly prevented the loss of cell viability and inhibited cell apoptosis induced by UVB in RCE cells. LBPs also inhibited UVB-induced loss of mitochondrial membrane potential, downregulation of Bcl-2, and upregulation of Bax and caspase-3. Finally, LBPs attenuated the phosphorylation of c-Jun NH2-terminal kinase (JNK triggered by UVB. In summary, LBPs protect RCE cells against UVB-induced damage and apoptosis, and the underlying mechanism involves the attenuation of the mitochondrial apoptosis pathway and the inhibition of JNK phosphorylation.

Endothelial induced EMT in breast epithelial cells with stem cell properties

DEFF Research Database (Denmark)

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

2011-01-01

endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D......492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close...

Plasticity between Epithelial and Mesenchymal States Unlinks EMT from Metastasis-Enhancing Stem Cell Capacity

NARCIS (Netherlands)

Beerling, Evelyne; Seinstra, Daniëlle; de Wit, Elzo; Kester, Lennart; van der Velden, Daphne; Maynard, Carrie; Schäfer, Ronny; van Diest, Paul; Voest, Emile; van Oudenaarden, Alexander; Vrisekoop, Nienke; van Rheenen, Jacco

2016-01-01

Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT) has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells

Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface.

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Chang-Lin, Joan-En; Kim, Kwang-Jin; Lee, Vincent H L

2005-06-01

Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits. Primary cultured RCrECL were grown at an air-interface on Clear-Snapwells precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na(+) and Cl(-) transport to overall short-circuit current (I(sc)) was evaluated by removing Na(+) and Cl(-), respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na(+) and Cl(-) using (22)Na and (36)Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na(+)-channel activities to net Na(+) fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl(2) and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl(-)-channel, and basolateral K(+)-channel and Na(+)(K(+))2Cl(-)-cotransporter activities, respectively, in active ion transport across RCrECL. I(sc) of RCrECL derived from pigmented rabbits was comprised of 64+/-2% and 44+/-5% for active Na(+) and Cl(-) transport, respectively, consistent with net Na(+) absorption and Cl(-) secretion of 0.062+/-0.006 and 0.046+/-0.008 muEq/cm(2)/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited I(sc) by up to approximately 50% with an IC(50) of 1 and 0.1 microm, respectively, consistent with participation of apical epithelial Na(+)-channels to net Na(+) absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl(2) to the basolateral and bumetanide to basolateral fluid decreased I(sc) by 86+/-1.5%, 53+/-3%, 18+/-1.8% and 13+/-1.9% in RCr

Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.

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Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X

2004-08-01

To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces

Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells

Science.gov (United States)

Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.

2009-01-01

PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis

Ultrastructural analysis of corneal exposure to UV radiation

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Pitts, D.G.; Bergmanson, J.P.G.; Chu, L.W.-F.

1987-01-01

The primate cornea was exposed to 300 nm UVR with five levels of radiant expsure from 0.08 to 0.6 Jcm/sup -2/. All cellular layers of the cornea were damaged at the 0.08 Jcm/sup -2/ exposure, and damage became more severe as the exposure level was increased. The corneal cells showed variable response in that essentially normal cells were found among damaged cells. Eight days post-exposure using the 0.6 Jcm/sup -2/ level, the epithelium had regained its normal thickness and was populated largely by normal appearing cells; however, the stroma showed damaged keratocytes and the loss of keratocytes. The corneal basement membranes (the epithelial basement membrane and the posterior limiting lamina) and the anterior limiting lamina were not damaged at any exposure level except for an isolated area along the epithelial basement membrane in one cornea. Therefore, one is lead to conclude that basement membranes are unaffected by UVR. The endothelium continued to demonstrate the loss of mitochondria, endoplasmic reticulum and some vacuoles at 8 days after exposure. However, the endothelium appeared to have resumed its physiological function as demonstrated by the reduced stromal oedema. This research gives the first complete description of UV-B induced corneal damage and repair of the full, in-depth cornea of the primate using the EM.

Ultrastructural analysis of corneal exposure to UV radiation

International Nuclear Information System (INIS)

Pitts, D.G.; Bergmanson, J.P.G.; Chu, L. W-F.

1987-01-01

The primate cornea was exposed to 300 nm UVR with five levels of radiant expsure from 0.08 to 0.6 Jcm -2 . All cellular layers of the cornea were damaged at the 0.08 Jcm -2 exposure, and damage became more severe as the exposure level was increased. The corneal cells showed variable response in that essentially normal cells were found among damaged cells. Eight days post-exposure using the 0.6 Jcm -2 level, the epithelium had regained its normal thickness and was populated largely by normal appearing cells; however, the stroma showed damaged keratocytes and the loss of keratocytes. The corneal basement membranes (the epithelial basement membrane and the posterior limiting lamina) and the anterior limiting lamina were not damaged at any exposure level except for an isolated area along the epithelial basement membrane in one cornea. Therefore, one is lead to conclude that basement membranes are unaffected by UVR. The endothelium continued to demonstrate the loss of mitochondria, endoplasmic reticulum and some vacuoles at 8 days after exposure. However, the endothelium appeared to have resumed its physiological function as demonstrated by the reduced stromal oedema. This research gives the first complete description of UV-B induced corneal damage and repair of the full, in-depth cornea of the primate using the EM. (author)

Minocycline inhibits alkali burn-induced corneal neovascularization in mice.

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Ou Xiao

Full Text Available The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV. A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS or minocycline twice a day (60 mg/kg or 30 mg/kg for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs. The mRNA expression levels of vascular endothelial growth factor (VEGF and its receptors (VEGFRs, basic fibroblast growth factor (bFGF, matrix metalloproteinases (MMPs, interleukin-1α, 1β, 6 (IL-1α, IL-1β, IL-6 were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1β and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1β, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1β, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d. can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.

Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

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Lough, Denver M; Wetter, Nathan; Madsen, Christopher; Reichensperger, Joel; Cosenza, Nicole; Cox, Lisa; Harrison, Carrie; Neumeister, Michael W

2016-02-01

Recent literature has shown that full-thickness wounds, devoid of the stem cell niche, can subsequently be reconstructed with functional skin elements following migration of the LGR6 epithelial stem cell into the wound bed. In this study, the authors use a variety of LGR6 epithelial stem cell-seeded scaffolds to determine therapeutic utility and regenerative potential in the immediate reconstruction of full-thickness wounds. Isolated LGR6 epithelial stem cells were seeded onto a spectrum of acellular matrices and monitored in both in vitro and in vivo settings to determine their relative capacity to regenerate tissues and heal wounds. Wound beds containing LGR6 stem cell-seeded scaffolds showed significantly augmented rates of healing, epithelialization, and hair growth compared with controls. Gene and proteomic expression studies indicate that LGR6 stem cell-seeded constructs up-regulate WNT, epidermal growth factor, and angiogenesis pathways. Finally, the addition of stromal vascular fraction to LGR6 stem cell-seeded constructs induces polarized tissue formation, nascent hair growth, and angiogenesis within wounds. LGR6 stem cells are able to undergo proliferation, differentiation, and migration following seeding onto a variety of collagen-based scaffolding. In addition, deployment of these constructs induces epithelialization, hair growth, and angiogenesis within wound beds. The addition of stromal vascular fraction to LGR6 stem cell-containing scaffolds initiated an early form of tissue polarization, providing for the first time a clinically applicable stem cell-based construct that is capable of the repair of full-thickness wounds and hair regeneration. Therapeutic, V.

Study on the establishment of corneal alkali chemical injury on rats

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Nan Hu

2013-06-01

Full Text Available AIM:To investigate the appropriate methods to establish corneal alkali chemical injury on rats. METHODS:The rats(n=87were randomly divided into three groups. Corneal alkali injury was induced by placing 1mol/L NaOH soaked filter paper on the limbus of right cornea for 20 seconds(group A, n=34or 40 seconds(group B, n=23, and on the central axis of the right cornea for 40 seconds(group C, n=30respectively. Corneal transparency, corneal ulceration, and corneal neovascularization were observed and recorded under slit- lamp biomicroscope on day 7 post-operation. RESULTS: Incidence of corneal ulceration, corneal perforation and positive rate of corneal fluorescein staining in limbal corneal injury groups(group A and Bwere significantly higher than that of central corneal injury group(group C(P<0.05. Incidence of corneal ulceration and corneal perforation in group B was significantly higher than group A(P<0.05. Corneal neovascularization was observed in all three groups. CONCLUSION: Corneal alkali burns induced by 3mm diameter central cornea injury are fit for the study of corneal neovascularization, while those induced by limbus injury for 20 seconds are fit for the study on limbal stem cells deficiency.

Corneal wound healing promoted by 3 blood derivatives: an in vitro and in vivo comparative study.

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Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Hernáez-Moya, Raquel; Durán, Juan A; Morales, María-Celia

2014-06-01

The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]. Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment. In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment. All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.

Epidermal growth factor in alkali-burned corneal epithelial wound healing.

Science.gov (United States)

Singh, G; Foster, C S

1987-06-15

We conducted a double-masked study to evaluate the effect of epidermal growth factor on epithelial wound healing and recurrent erosions in alkali-burned rabbit corneas. Epithelial wounds 10 mm in diameter healed completely under the influence of topical epidermal growth factor, whereas the control corneas did not resurface in the center. On reversal of treatment, the previously nonhealing epithelial defects healed when treated with topical epidermal growth factor eyedrops. Conversely, the epidermal growth factor-treated and resurfaced corneas developed epithelial defects when treatment was discontinued. Histopathologic examination disclosed hyperplastic epithelium growing over the damaged stroma laden with polymorphonuclear leukocytes when treated with epidermal growth factor eyedrops, but it did not adhere to the underlying tissue. Hydropic changes were seen intracellularly as well as between the epithelial cells and the stroma.

Regulation of Pituitary Stem Cells by Epithelial to Mesenchymal Transition Events and Signaling Pathways

Science.gov (United States)

Cheung, Leonard Y. M.; Davis, Shannon W.; Brinkmeier, Michelle L.; Camper, Sally A.; Pérez-Millán, María Inés

2017-01-01

The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized cells arise from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. The adult anterior pituitary responds to physiological challenge by mobilizing the SOX2-expressing progenitor pool and producing additional hormone-producing cells. Knowledge of the role of signaling pathways and extracellular matrix components in these processes may lead to improvements in the efficiency of differentiation of embryonic stem cells or induced pluripotent stem cells into hormone producing cells in vitro. Advances in our basic understanding of pituitary stem cell regulation and differentiation may lead to improved diagnosis and treatment for patients with hypopituitarism. PMID:27650955

Epithelial cells as alternative human biomatrices for comet assay.

Science.gov (United States)

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

Illustration of extensive extracellular matrix at the epithelial-mesenchymal interface within the renal stem/progenitor cell niche

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Minuth Will W

2012-09-01

Full Text Available Abstract Background Stem/progenitor cells are promising candidates to treat diseased renal parenchyma. However, implanted stem/progenitor cells are exposed to a harmful atmosphere of degenerating parenchyma. To minimize hampering effects after an implantation investigations are in progress to administer these cells within an artificial polyester interstitum supporting survival. Learning from nature the renal stem/progenitor cell niche appears as a valuable model. At this site epithelial stem/progenitor cells within the collecting duct ampulla face mesenchymal stem/progenitor cells. Both cell types do not have close contact but are separated by a wide interstitium. Methods To analyze extracellular matrix in this particular interstitium, special contrasting for transmission electron microscopy was performed. Kidneys of neonatal rabbits were fixed in solutions containing glutaraldehyde (GA or in combination with cupromeronic blue, ruthenium red and tannic acid. Results GA revealed a basal lamina at the ampulla and a bright but inconspicuously looking interstitial space. In contrast, GA containing cupromeronic blue exhibits numerous proteoglycan braces lining from the ampulla towards the interstitial space. GA containing ruthenium red or tannic acid demonstrates clouds of extracellular matrix protruding from the basal lamina of the ampulla to the surface of mesenchymal stem/progenitor cells. Conclusions The actual data show that the interstitium between epithelial and mesenchymal stem/progenitor cells contains much more and up to date unknown extracellular matrix than earlier observed by classical GA fixation.

Recovery of Corneal Sensitivity and Increase in Nerve Density and Wound Healing in Diabetic Mice After PEDF Plus DHA Treatment.

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He, Jiucheng; Pham, Thang Luong; Kakazu, Azucena; Bazan, Haydee E P

2017-09-01

Diabetic keratopathy decreases corneal sensation and tear secretion and delays wound healing after injury. In the current study, we tested the effect of treatment with pigment epithelium-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) on corneal nerve regeneration in a mouse model of diabetes with or without corneal injury. The study was performed in streptozotocin-induced diabetic mice (C57BL/6). Ten weeks after streptozotocin injection, diabetic mice showed significant decreases of corneal sensitivity, tear production, and epithelial subbasal nerve density when compared with age-matched normal mice. After diabetic mice were wounded in the right eye and treated in both eyes with PEDF+DHA for 2 weeks, there was a significant increase in corneal epithelial nerve regeneration and substance P-positive nerve density in both wounded and unwounded eyes compared with vehicle-treated corneas. There also was elevated corneal sensitivity and tear production in the treated corneas compared with vehicle. In addition, PEDF+DHA accelerated corneal wound healing, selectively recruited type 2 macrophages, and prevented neutrophil infiltration in diabetic wounded corneas. These results suggest that topical treatment with PEDF+DHA promotes corneal nerve regeneration and wound healing in diabetic mice and could potentially be exploited as a therapeutic option for the treatment of diabetic keratopathy. © 2017 by the American Diabetes Association.

Assessment of Corneal and Tear Film Parameters in Rheumatoid Arthritis Patients Using Anterior Segment Spectral Domain Optical Coherence Tomography.

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El-Fayoumi, Dina; Youssef, Maha Mohamed; Khafagy, Mohamed Mahmoud; Badr El Dine, Nashwa; Gaber, Wafaa

2018-01-01

To study the corneal changes in rheumatoid arthritis (RA) patients in vivo, using spectral domain anterior segment optical coherence tomography (AS-OCT). A case-control study was done on 43 RA patients and 40 controls. The disease activity score (DAS28-ESR) was calculated and all participants had lower tear meniscus, corneal thickness, and epithelial thickness evaluation using AS-OCT. The lower tear meniscus height (LTMH) and the lower tear meniscus area (LTMA) were significantly lower in the RA patients than in controls (p < 0.001). RA patients also had a significantly thinner central corneal thickness (p = 0.02) and their epithelium was found to be thinner in the superotemporal peripheral sector. The LTMH and LTMA are significantly reduced in RA patients, despite the absence of clinical diagnosis of dry eye. RA patients have thinner corneal thickness and epithelial thickness than controls, which did not correlate with either disease duration or activity.

Delayed healing of corneal epithelium after phototherapeutic keratectomy for lattice dystrophy.

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Das, Sujata; Langenbucher, Achim; Seitz, Berthold

2005-04-01

To evaluate the time period necessary for complete epithelial healing after phototherapeutic keratectomy (o-PTK) carried out for various superficial corneal opacities. A total of 197 eyes were divided into 9 groups: group 1, Cogan dystrophy including recurrences (n = 15); group 2, Reis Bucklers dystrophy including recurrences (n = 12); group 3, granular dystrophy including recurrences (n = 63); group 4, lattice dystrophy including recurrences (n = 19); group 5, macular dystrophy including recurrences (n = 10); group 6, herpetic scars (n = 5); group 7, corneal scars of nonherpetic origin (including scrofulous, traumatic, central keratoconus, post-pterygium surgery) (n = 31); group 8, Salzmann nodular degeneration (n = 22); and group 9, miscellaneous (such as bullous keratopathy, acute chemical burn, corneal degeneration) (n = 20). After o-PTK, patients were examined daily at the slit lamp using fluorescein and blue light. The time period necessary for complete healing of the epithelial defect was compared among these groups. Delayed healing was considered where the epithelium was not closed after 7 days. One hundred sixty-one eyes (95%) healed within 7 days. Overall, 63%, 80%, and 85% of epithelial defects were closed within 3, 4, and 5 days, respectively. Out of 9 eyes that had delayed healing, 6 eyes (67%) belonged to lattice dystrophy category. Mean time taken for healing in group 4 (8.6 +/- 8.4 days) was significantly longer than those in group 1 (3.0 +/- 1.5 days, P = 0.009), group 2 (3.7 +/- 3.1 days, P = 0.03), group 3 (3.1 +/- 1.5 days, P = 0.001), group 5 (2.7 +/- 0.8 days, P = 0.01), group 7 (3.6 +/- 2.4 days, P = 0.007), group 8 (3.3 +/- 1.3 days, P = 0.009), and group 9 (3.0 +/- 1.9 days, P = 0.011). Eyes with lattice corneal dystrophy suffered from delayed epithelial healing after o-PTK. In addition to adequate counseling, these patients should be followed up closely until complete closure of the epithelium to avoid ulceration, scarring, or even

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT)-Phenotypic Cells: Are They Cousins or Twins?

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Kong, Dejuan; Li, Yiwei; Wang, Zhiwei; Sarkar, Fazlul H.

2011-01-01

Cancer stem cells (CSCs) are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT), induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT-Phenotypic Cells: Are They Cousins or Twins?

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Fazlul H. Sarkar

2011-02-01

Full Text Available Cancer stem cells (CSCs are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT, induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors.

Comparative Confocal and Histopathological Study of Corneal Changes in Multiple Myeloma.

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Micali, Antonio; Roszkowska, Anna M; Postorino, Elisa I; Rania, Laura; Aragona, Emanuela; Wylegala, Edward; Nowinska, Anna; Ieni, Antonio; Calimeri, Sebastiano; Pisani, Antonina; Aragona, Pasquale; Puzzolo, Domenico

2017-01-01

Corneal opacities rarely occur in multiple myeloma (MM). Our study correlates the findings of in vivo confocal microscopy (IVCM), a useful diagnostic tool, with histopathological features of corneal opacities appearing in a patient with MM. Case report. A 53-year-old man developed corneal opacities in both eyes, more pronounced in the left eye. After IVCM examination, he underwent penetrating keratoplasty in the left eye, and the button was processed for light and electron microscopy and immunohistochemistry. The diagnosis of MM was made, as confirmed by the elevation of IgGk light chains. IVCM demonstrated hyperreflective areas at the epithelial level, hyperreflective keratocytes of dendritic and lamellar morphology in whole stroma, and hyperreflective endothelial cells. Histopathological examination disclosed many vacuoles in the epithelial cell cytoplasm and a homogenous granular material in the Bowman layer. In stroma, keratocytes of different shape and size, with vesicles laden with an abnormal material, were evident. In Descemet membrane, the posterior nonbanded zone had a honeycomb appearance because of the presence of many roundish spaces among wide-spaced collagen fibers. Endothelial cells demonstrated vesicles filled with a material of uneven electron density. Immunohistochemical analysis showed strong positivity for IgGk light chains in keratocytes and among stromal lamellae. This is the first study describing a correspondence between IVCM features and histopathological alterations observed in corneal opacities in MM. The results of this study improve the current understanding of the pictures obtained by IVCM studies.

Inhibitory effects of regorafenib, a multiple tyrosine kinase inhibitor, on corneal neovascularization

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Halil Ibrahim Onder

2014-04-01

Full Text Available AIM:To evaluate the inhibitory effects of regorafenib (BAY 73-4506, a multikinase inhibitor, on corneal neovascularization (NV.METHODS:Thirty adult male Sprague-Dawley rats weighing 250-300 g, were used. Corneal NV was induced by NaOH in the left eyes of each rat. Following the establishment of alkali burn, the animals were randomized into five groups according to topical treatment. Group 1 (n = 6 received 0.9% NaCl, Group 2 (n = 6 received dimethyl sulfoxide, Group 3 (n = 6 received regorafenib 1 mg/mL, Group 4 (n =6 received bevacizumab 5 mg/mL and Group 5 (n = 6 received 0.1% dexamethasone phosphate. On the 7d, the corneal surface covered with neovascular vessels was measured on photographs as the percentage of the cornea’s total area using computer-imaging analysis. The corneas obtained from rats were semiquantitatively evaluated for caspase-3 and vascular endothelial growth factor by immunostaining.RESULTS:A statistically significant difference in the percent area of corneal NV was found among the groups (P <0.001. Although the Group 5 had the smallest percent area of corneal NV, there was no difference among Groups 3, 4 and 5 (P >0.005. There was a statistically significant difference among the groups in apoptotic cell density (P = 0.002. The staining intensity of vascular endothelial growth factor in the epithelial and endothelial layers of cornea was significantly different among the groups (P <0.05. The staining intensity of epithelial and endothelial vascular endothelial growth factor was significantly weaker in Groups 3, 4 and 5 than in Groups 1 and 2.CONCLUSION: Topical administration of regorafenib 1 mg/mL is partly effective for preventing alkali-induced corneal NV in rats.

Three-dimensional epithelial tissues generated from human embryonic stem cells.

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Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

2009-11-01

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

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Mandayam O. Nandan

2015-01-01

Full Text Available Krüppel-like factor 5 (KLF5 is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5 has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5fl/fl were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112 days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC cells that express Lgr5. By 11 days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14 days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112 days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells.

Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

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Claire M Elkins

Full Text Available Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS, borate buffered saline (BBS, or Sensitive Eyes Plus Saline Solution (Sensitive Eyes, either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

The Palisades of Vogt in Congenital Corneal Opacification (An American Ophthalmological Society Thesis).

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Nischal, Ken K; Lathrop, Kira L

2016-08-01

The purposes of this study are first, to determine if the palisades of Vogt (POV) are present or absent in cases of congenital corneal opacification (CCO) by using spectral domain ocular coherence tomography (SD-OCT), and second, in those cases already undergoing penetrating keratoplasty (PKP), to see whether the absence or presence of POV corresponds to re-epithelialization following transplant. This was a retrospective case review of 20 eyes (10 normal, 10 with CCO) evaluated with SD-OCT. The operator was masked to the clinician's assessment of the ocular surface. In those cases where the decision to perform PKP had already been made, the correlation between POV presence or absence and posttransplant graft epithelialization was determined. All cases were imaged without adverse event. Nine eyes showed some evidence of POV and corresponding vasculature. Eight of 10 affected eyes underwent PKP, and subsequently 7 eyes epithelialized and 2 showed some peripheral neovascularization. The one eye that showed no signs of POV was the one that failed to epithelialize. All control subjects had consistent and regular POV. Congenital corneal opacification is rare, and this study shows that at least some POV are present in the majority of cases of CCO. However, the palisades may not be entirely normal compared to age-matched controls. When there was absence of POV in a case of CCO, there was immediate and complete failure of epithelialization.

Epithelial-to-mesenchymal plasticity of cancer stem cells: therapeutic targets in hepatocellular carcinoma

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Aparna Jayachandran

2016-08-01

Full Text Available Abstract Hepatocellular carcinoma (HCC remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future.

The Supportive Role of Insulin-Like Growth Factor-I in the Differentiation of Murine Mesenchymal Stem Cells into Corneal-Like Cells

Czech Academy of Sciences Publication Activity Database

Trošan, Peter; Javorková, Eliška; Zajícová, Alena; Hájková, Michaela; Heřmánková, Barbora; Kössl, Jan; Holáň, Vladimír

2016-01-01

Roč. 7, č. 17 (2016), s. 23156-23169 ISSN 1547-3287 R&D Projects: GA ČR(CZ) GA14-12580S; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1309; GA MŠk(CZ) LO1508 Institutional support: RVO:68378041 Keywords : mesenchymal stem cells * corneal-like cells * insulin -like growth factor-I * differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.562, year: 2016

Correlation between epithelial thickness in normal corneas, untreated ectatic corneas, and ectatic corneas previously treated with CXL; is overall epithelial thickness a very early ectasia prognostic factor?

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Kanellopoulos AJ

2012-05-01

Full Text Available Anastasios John Kanellopoulos,1,2 Ioannis M Aslanides,3 George Asimellis11Laservision Eye Institute, Athens, 2Emmetropia Mediterranean Eye Clinic, Crete, Greece, 3New York University School of Medicine, NY, USAPurpose: To determine and correlate epithelial corneal thickness (pachymetric measurements taken with a digital arc scanning very high frequency ultrasound biomicroscopy (HF UBM imaging system (Artemis-II, and compare mean and central epithelial thickness among normal eyes, untreated keratoconic eyes, and keratoconic eyes previously treated with collagen crosslinking (CXL.Methods: Epithelial pachymetry measurements (topographic mapping were conducted on 100 subjects via HF UBM. Three groups of patients were included: patients with normal eyes (controls, patients with untreated keratoconic eyes, and patients with keratoconic eyes treated with CXL. Central, mean, and peripheral corneal epithelial thickness was examined for each group, and a statistical study was conducted.Results: Mean, central, and peripheral corneal epithelial thickness was compared between the three groups of patients. Epithelium thickness varied substantially in the keratoconic group, and in some cases there was a difference of up to 20 µm between various points of the same eye, and often a thinner epithelium coincided with a thinner cornea. However, on average, data from the keratoconic group suggested an overall thickening of the epithelium, particularly over the pupil center of the order of +3 µm, while the mean epithelium thickness was on average +1.1 µm, compared to the control population (P = 0.005. This overall thickening was more pronounced in younger patients in the keratoconic group. Keratoconic eyes previously treated with CXL showed, on average, virtually the same average epithelium thickness (mean –0.7 µm, –0.2 µm over the pupil center, –0.9 µm over the peripheral zone as the control group. This finding further reinforces our novel theory of the

Hedgehog Signaling Regulates Epithelial-Mesenchymal Transition in Pancreatic Cancer Stem-Like Cells

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Wang, Feng; Ma, Ling; Zhang, Zhengkui; Liu, Xiaoran; Gao, Hongqiao; Zhuang, Yan; Yang, Pei; Kornmann, Marko; Tian, Xiaodong; Yang, Yinmo

2016-01-01

Hedgehog (Hh) signaling is crucially involved in tumorigenesis. This study aimed to assess the role of Hh signaling in the regulation of epithelial-mesenchymal transition (EMT), stemness properties and chemoresistance of human pancreatic Panc-1 cancer stem cells (CSCs). Panc-1 cells were transfected with recombinant lentiviral vectors to silence SMO and serum-free floating-culture system was used to isolate Panc-1 tumorspheres. The expression of CSC and EMT markers was detected by flow cytometry, real-time RT-PCR and Western blot analysis. Malignant behaviors of Panc-1 CSC were evaluated by tumorigenicity assays and nude mouse lung metastasis model. We found that tumorspheres derived from pancreatic cancer cell line Panc-1 possessed self-renewal, differentiation and stemness properties. Hh pathway and EMT were active in Panc-1 tumorspheres. Inhibition of Hh signaling by SMO knockdown inhibited self-renewal, EMT, invasion, chemoresistance, pulmonary metastasis, tumorigenesis of pancreatic CSCs. In conclusion, Hh signaling contributes to the maintenance of stem-like properties and chemoresistance of pancreatic CSC and promotes the tumorigenesis and metastasis of pancreatic cancer. Hh pathway is a potential molecular target for the development of therapeutic strategies for pancreatic CSCs. PMID:26918054

ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

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Jarom Heijmans

2013-04-01

Full Text Available Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER stress and activity of the unfolded protein response (UPR are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

The theory and art of corneal cross-linking.

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McQuaid, Rebecca; Cummings, Arthur B; Mrochen, Michael

2013-08-01

Before the discovery of corneal cross-linking (CXL), patients with keratoconus would have had to undergo corneal transplantation, or wear rigid gas permeable lenses (RGPs) that would temporarily flatten the cone, thereby improving the vision. The RGP contact lens (CL) would not however alter the corneal stability and if the keratoconus was progressive, the continued steepening of the cone would occur under the RGP CL. To date, the Siena Eye has been the largest study to investigate long term effects of standard CXL. Three hundred and sixty-three eyes were treated and monitored over 4 years, producing reliable long-term results proving long-term stability of the cornea by halting the progression of keratoconus, and proving the safety of the procedure. Traditionally, CXL requires epithelial removal prior to corneal soakage of a dextran-based 0.1% riboflavin solution, followed by exposure of ultraviolet-A (UV-A) light for 30 min with an intensity of 3 mW/cm2. A series of in vitro investigations on human and porcine corneas examined the best treatment parameters for standard CXL, such as riboflavin concentration, intensity, wavelength of UV-A light, and duration of treatment. Photochemically, CXL is achieved by the generation of chemical bonds within the corneal stroma through localized photopolymerization, strengthening the cornea whilst minimizing exposure to the surrounding structures of the eye. In vitro studies have shown that CXL has an effect on the biomechanical properties of the cornea, with an increased corneal rigidity of approximately 70%. This is a result of the creation of new chemical bonds within the stroma.

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

OpenAIRE

Foltz, LP; Clegg, DO

2017-01-01

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embry...

Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

Science.gov (United States)

Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

2013-11-01

Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

International Nuclear Information System (INIS)

Katano, Takahito; Ootani, Akifumi; Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi; Toda, Shuji; Joh, Takashi

2013-01-01

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

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Katano, Takahito [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Ootani, Akifumi [Department of Gastroenterology and GI Endoscopy Center, Shin-Kokura Hospital, Federation of National Public Service Personnel Mutual Aid Associations, 1-3-1 Kanada, Kokurakita-ku, Kitakyushu 803-0816 (Japan); Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Mizoshita, Tsutomu, E-mail: tmizoshi@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Toda, Shuji [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

2013-03-22

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

Мodification of Mechanical Limbal Stem Cell Deficiency Model

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A. V. Bezushko

2018-01-01

Full Text Available Introduction. Ocular surface diseases related with limbal epithelial stem cells dysfunction were united in term “limbal stem cell  deficiency” (LSCD. For experimental study of the regenerative processes and evaluation of the success of new LSCD treatingmethods LSCD model is required. Various LSCD models were proposed in the experiment to study: mechanical, thermal, chemical,medicamental. The main lack of these models were the relative high cost and complexity of execution. The mechanical model allows forthe guaranteed removal of tissues containing LESCs, and therefore seems to be the most acceptable. We offered a modification of themechanical LSCD model in rabbits. Purpose to create a standardized modification of the mechanical limbal stem cell deficiency modelin the experiment. Material and methods. The experimental study was performed in 10 mature Chinchilla rabbits (20 eyes with anaverage weight 2.5–3.5 kg. With the local anesthesia, after a 40-second application of the filter paper impregnated with 20% ethanol,the corneal epithelium was removed. With microsurgical diamond blade we metered limb portion of 4 mm width, 0.2 mm deep, andit was removed along the 360° rim. Results. On the 30th day we discovered corneal opacity and neovascularization with conjunctivalpannus extending to the optical zone of the cornea. Histological examination revealed tissue edema, inflammatory infiltration, andnewly formed vessels. In some places, thinning of epithelium to one row of flattened cells was observed. The Bowman membrane wasdeformed and practically not detected. Histological examination and impression cytology confirmed the presence of goblet cells in thecorneal epithelium. Conclusions. Our modification of the mechanical limbal stem cell deficiency model is devoid of the main lacks ofprevious models, such as the high cost and complexity of execution, provides intraoperative limbal tissue resection depth control andexcludes the possibility of the

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2.

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Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X

2008-05-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappaB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappaB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules.

Comparison of the effects of ophthalmic solutions on human corneal epithelial cells using fluorescent dyes.

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Xu, Manlong; Sivak, Jacob G; McCanna, David J

2013-11-01

To investigate the effect of differently preserved ophthalmic solutions on the viability and barrier function of human corneal epithelial cells (HCEC) using fluorescent dyes. HCEC monolayers were exposed to the ophthalmic solutions containing benzalkonium chloride (BAK), edetate disodium, polyquad, stabilized oxychloro complex (Purite), sodium perborate, or sorbic acid for 5 min, 15 min, and 1 h. At 24 h after exposure, the cultures were assessed for metabolic activity using alamarBlue. The enzyme activity, membrane integrity, and apoptosis were evaluated using confocal microscopy. Barrier function was assessed using sodium fluorescein. The metabolic assay showed that the BAK-preserved ophthalmic solutions significantly reduced cell viability after a 5-min exposure compared to the phosphate buffered saline treated control (POphthalmic solutions with new preservatives had varying time-dependent adverse effects on cell viability, and the preservative-free solution had the least effect on HCEC. Sodium fluorescein permeability showed that HCEC monolayers treated with BAK-preserved solutions were more permeable to sodium fluorescein than those treated by the other ophthalmic solutions (Psolutions had greater adverse effects on metabolic activity, enzyme activity, membrane integrity, cell viability, and barrier function than the solutions that were not preserved with BAK. Our study suggests that BAK-free especially, preservative-free ophthalmic solutions are safer alternatives to BAK-preserved ones.

Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells

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Hombauer, H; Minguell, J J

2000-01-01

This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high...

A Case of Medication-Resistant Acanthamoeba Keratitis Treated by Corneal Crosslinking in Turkey

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Goktug Demirci

2013-01-01

Full Text Available Purpose. To report a case of medication-resistant acanthamoeba keratitis (AK treated successfully by corneal crosslinking (CXL. Methods. A 26-year-old male with medication-resistant AK underwent a standard CXL procedure with local anesthesia, followed by central corneal epithelial debridement, application of riboflavin 0.1%, and UV-A irradiation. Results. The patient experienced a dramatic symptomatic improvement within 24 hours. At two months, keratitis was healed with a semitransparent paracentral scar that did not affect visual acuity. Conclusions. Our experience, considered in the context of recent studies, suggests that CXL may be an option for selected patients with medication-resistant AK and corneal melting. CXL allows patients to avoid emergency keratoplasty and experience rapid symptomatic relief.

The theory and art of corneal cross-linking

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Rebecca McQuaid

2013-01-01

Full Text Available Before the discovery of corneal cross-linking (CXL, patients with keratoconus would have had to undergo corneal transplantation, or wear rigid gas permeable lenses (RGPs that would temporarily flatten the cone, thereby improving the vision. The RGP contact lens (CL would not however alter the corneal stability and if the keratoconus was progressive, the continued steepening of the cone would occur under the RGP CL. To date, the Siena Eye has been the largest study to investigate long term effects of standard CXL. Three hundred and sixty-three eyes were treated and monitored over 4 years, producing reliable long-term results proving long-term stability of the cornea by halting the progression of keratoconus, and proving the safety of the procedure. Traditionally, CXL requires epithelial removal prior to corneal soakage of a dextran-based 0.1% riboflavin solution, followed by exposure of ultraviolet-A (UV-A light for 30 min with an intensity of 3 mW/cm2. A series of in vitro investigations on human and porcine corneas examined the best treatment parameters for standard CXL, such as riboflavin concentration, intensity, wavelength of UV-A light, and duration of treatment. Photochemically, CXL is achieved by the generation of chemical bonds within the corneal stroma through localized photopolymerization, strengthening the cornea whilst minimizing exposure to the surrounding structures of the eye. In vitro studies have shown that CXL has an effect on the biomechanical properties of the cornea, with an increased corneal rigidity of approximately 70%. This is a result of the creation of new chemical bonds within the stroma.

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.

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Couture, Camille; Zaniolo, Karine; Carrier, Patrick; Lake, Jennifer; Patenaude, Julien; Germain, Lucie; Guérin, Sylvain L

2016-02-01

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Improving the mechanical properties of collagen-based membranes using silk fibroin for corneal tissue engineering.

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Long, Kai; Liu, Yang; Li, Weichang; Wang, Lin; Liu, Sa; Wang, Yingjun; Wang, Zhichong; Ren, Li

2015-03-01

Although collagen with outstanding biocompatibility has promising application in corneal tissue engineering, the mechanical properties of collagen-based scaffolds, especially suture retention strength, must be further improved to satisfy the requirements of clinical applications. This article describes a toughness reinforced collagen-based membrane using silk fibroin. The collagen-silk fibroin membranes based on collagen [silk fibroin (w/w) ratios of 100:5, 100:10, and 100:20] were prepared by using silk fibroin and cross-linking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. These membranes were analyzed by scanning electron microscopy and their optical property, and NaCl and tryptophan diffusivity had been tested. The water content was found to be dependent on the content of silk fibroin, and CS10 membrane (loading 10 wt % of silk fibroin) performed the optimal mechanical properties. Also the suture experiments have proved CS10 has high suture retention strength, which can be sutured in rabbit eyes integrally. Moreover, the composite membrane proved good biocompatibility for the proliferation of human corneal epithelial cells in vitro. Lamellar keratoplasty shows that CS10 membrane promoted complete epithelialization in 35 ± 5 days, and their transparency is restored quickly in the first month. Corneal rejection reaction, neovascularization, and keratoconus are not observed. The composite films show potential for use in the field of corneal tissue engineering. © 2014 Wiley Periodicals, Inc.

Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

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Hu, Wen-Yang; Hu, Dan-Ping; Xie, Lishi; Li, Ye; Majumdar, Shyama; Nonn, Larisa; Hu, Hong; Shioda, Toshi; Prins, Gail S

2017-08-01

Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

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Wen-Yang Hu

2017-08-01

Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium via a c-jun N-terminal kinase 2 (JNK2) pathway.

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Pelegrino, F S A; Pflugfelder, S C; De Paiva, C S

2012-01-01

Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1β, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of Pentacam on the posterior corneal surface height changes after LASEK and LASIK operation

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Qing-Song Zhang

2014-05-01

Full Text Available AIM: To explore the application of Pentacam excimer laser epithelial keratomileusis(LASEKand excimer laser in situ keratomileusis(LASIKafter the changes of posterior corneal surface height.METHODS: Retrospective analysis of clinical data of 100 patients with myopia by using LASEK and LASIK for the treatment of the 50 patients(100 eyesin our hospital from January 2013 to June 2013, surface height changes after preoperative and postoperative 3 months were compared by measuring Pentacam corneal analysis system.RESULTS: Three months after operation, the LASEK posterior corneal surface height was 7.4±5.0mm, significantly higher than 5.6±3.4mm before operation, LASIK posterior corneal surface height was 7.5±5.1mm, significantly higher than 5.5±3.5mm before operation, the differences were statistically significant(PP>0.05.CONCLUSION: LASEK and LASIK on corneal posterior surface forward, LASIK is slightly obvious in early period.

Plasma rich in growth factors (PRGF-Endoret) stimulates corneal wound healing and reduces haze formation after PRK surgery.

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Anitua, E; Muruzabal, F; Alcalde, I; Merayo-Lloves, J; Orive, G

2013-10-01

This study evaluated the efficacy of Plasma rich in growth factors (PRGF-Endoret) on the corneal wound healing process after Photorefractive keratectomy (PRK). To address this, blood from three healthy donors was collected, centrifuged and, the whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were collected. The effects of F3 and WP on the proliferation and migration of human corneal epithelial cells (HCE) were analyzed. PRK was performed on C57BL/6 mice. Animals were divided in three treatment groups: Control, F3, and WP. Corneal wound healing and haze formation were evaluated macroscopically. Eyes were collected at 1, 2, 3, and 7 days after surgery, and were processed for histological studies. Immunofluorescence was used to assess cellular proliferation, apoptosis and myofibroblast transformation in the mouse cornea. Results showed a significant increased on proliferation and wound healing after F3 and WP treatment when compared with control group. In vivo studies showed significant reduction on haze formation in mice treated with both PRGF-Endoret formulations (F3 and WP). Histological studies showed an increase of epithelial cell proliferation in corneas of control group, promoting an epithelial hyperplasia. The number of SMA-positive cells (corresponding to myofibroblast differentiation) was significantly lower in the PRGF-Endoret group than in the control group, correlating with the higher transparence results observed macroscopically in both PRGF-Endoret groups. According to this, it can be concluded that PRGF-Endoret accelerates corneal tissue regeneration after PRK, reducing haze formation. Copyright © 2013 Elsevier Ltd. All rights reserved.

In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

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Schmid, Richard; Tarau, Ioana-Sandra; Rossi, Angela; Leonhardt, Stefan; Schwarz, Thomas; Schuerlein, Sebastian; Lotz, Christian; Hansmann, Jan

2018-01-01

The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting. © 2017 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Outcome of cataract surgery following simple limbal epithelial transplantation for lime injury-induced limbal stem cell deficiency

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Nair, Dhanyasree

2015-01-01

A 19-year-old woman presented to us after being diagnosed elsewhere with right eye total limbal stem cell deficiency resulting from a lime burn. She was advised to undergo limbal stem cell transplantation, but failed to immediately do so. Two years later, she underwent cultivated limbal epithelial transplantation (CLET). As she had severe loss of vision with persisting conjunctival nodule and symblepharon 2 years following surgery, an impression of failed CLET was formed. Subsequently, simple limbal epithelial transplantation (SLET) was performed. Nine months later, she developed a cataract in her right eye; the cataract was extracted and posterior chamber intraocular lens implanted. The unaided visual acuity improved from light perception at presentation to 20/60 at 1-week postoperatively. At 5 months follow-up, the patient continued to maintain 20/60 visual acuity in her right eye. This case describes the outcome of cataract surgery following SLET, emphasising the need to perform cataract surgery in complicated cataracts for a better visual prognosis. PMID:26698204

Management of a Small Paracentral Corneal Perforation Using Iatrogenic Iris Incarceration and Tissue Adhesive

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Akira Kobayashi

2012-07-01

Full Text Available Background: Surgical intervention for corneal perforation is indicated when the anterior chamber does not reform within a short period of time. Herein, we report the successful management of a small paracentral corneal perforation using autologous iris incarceration and tissue adhesive. Case: A 41-year-old man developed a small paracentral corneal perforation (0.5 mm in size in the right eye, while the treating physician attempted to remove the residual rust ring after removal of a piece of metallic foreign body. Observations: The eye was initially managed with a bandage soft contact lens to ameliorate the aqueous leakage; however, without success. Iatrogenic iris incarceration of the wound was first induced, followed by application of cyanoacrylate tissue adhesive to the perforated site. As a result, the anterior chamber was immediately reformed and maintained. Complete corneal epithelialization of the perforation was achieved in 2 months without visual compromises. Conclusions: Cyanoacrylate tissue adhesive with iatrogenic incarceration of the autologous iris was effective in treating this type of small corneal perforation. This technique is simple and potentially useful for small paracentral corneal perforations outside the visual axis and without good apposition.

A new multiple noncontinuous puncture (pointage technique for corneal tattooing

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Jin Hyoung Park

2015-10-01

Full Text Available AIM:To assess the safety and cosmetic efficacy of a new multiple noncontinuous transepithelial puncture technique for tattooing a decompensated cornea.METHODS:It was anon-comparative clinical case series study.The study examines 33 eyes in 33 patients with total corneal opacity due to corneal decompensation, which developed following intraocular surgery.Corneal tattooing was performed using the multiple noncontinuous transepithelial puncture technique (i.e. pointage. The safety of this new surgical strategy was assessed by occurrence of adverse events for the follow-up period. The cosmetic efficacy was determined by the patient’s cosmetic satisfaction and independent observer’s opinion about patient appearance.RESULTS:Seven women and 26 men were included in the study. The mean age was 46.4±17.5y (range:7-67. In total, 30 of 33 patients (91% reported cosmetic satisfaction within the follow-up period. Only 3 patients (9% required additional tattooing due to cosmetic unsatisfaction. Cosmetic outcomes were analyzed and classified as excellent or good in 13 (39% and 17 (52% patients, respectively. No serious adverse events developed, except delayed epithelial healing in 3 cases.CONCLUSION:The cosmetic outcomes of the multiple noncontinuous transepithelial puncture technique for corneal tattooing were good. The safety of this method is higher than conventional procedures. This new procedure also provides improved cost-effectiveness and safety over current corneal tattooing techniques.

Area and depth of surfactant-induced corneal injury predicts extent of subsequent ocular responses.

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Jester, J V; Petroll, W M; Bean, J; Parker, R D; Carr, G J; Cavanagh, H D; Maurer, J K

1998-12-01

To correlate area and depth of initial corneal injury induced by surfactants of differing type and irritant properties with corneal responses and outcome in the same animals over time by using in vivo confocal microscopy (CM). Six groups of six adult rabbits were treated with anionic, cationic, and nonionic surfactants that caused different levels of ocular irritation. Test materials included slight irritants: 5% sodium lauryl sulfate (SLS), polyoxyethylene glycol monoalkyl ether (POE), and 5% 3-isotridecyloxypropyl-bis(polyoxyethylene) ammonium chloride (ITDOP); mild irritants: 5% 3-decyloxypropyl-bis(polyoxyethylene) amine (DOP) and sodium linear alkylbenzene sulfonate (LAS); and a moderate irritant: a proprietary detergent (DTRGT). Ten microliters surfactant were directly applied to the cornea of one eye of each rabbit. Ten untreated rabbits served as control subjects. Area and depth of initial injury was determined by using in vivo CM to measure epithelial thickness, epithelial cell size, corneal thickness, and depth of stromal injury in four corneal regions at 3 hours and at day 1. Area and depth of corneal responses to injury were evaluated at various times from days 3 through 35 by macroscopic grading and quantitative confocal microscopy through-focusing (CMTF). In vivo CM revealed corneal injury with slight irritants to be restricted to the epithelium, whereas the mild and moderate irritants caused complete epithelial cell loss with increasing anterior stromal damage: DOP < LAS < DTRGT. With the slight ocular irritants there was little or no change in corneal thickness or the CMTF intensity profiles. Three hours after treatment, mild and moderate ocular irritants caused a significant increase in corneal thickness, which peaked at day 1 with DOP (483.3+/-80.1 microm) and LAS (572.3+/-60.0 microm) and day 3 with DTRGT (601.4+/-68.7 microm); returning to normal (similar to control values) by day 7 with DOP and day 35 with LAS and DTRGT. The CMTF intensity

Acquisition cancer stemness, mesenchymal transdifferentiation, and chemoresistance properties by chronic exposure of oral epithelial cells to arecoline.

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Wang, Tung Yuan; Peng, Chih-Yu; Lee, Shiuan-Shinn; Chou, Ming-Yung; Yu, Cheng-Chia; Chang, Yu-Chao

2016-12-20

Oral squamous cell carcinoma (OSCC), one of the most deadliest malignancies in the world, is caused primarily by areca nut chewing in Southeast Asia. The mechanisms by which areca nut participates in OSCC tumorigenesis are not well understood. In this study, we investigated the effects of low dose long-term arecoline (10 μg/mL, 90-days), a major areca nut alkaloid, on enhancement cancer stemness of human oral epithelial (OE) cells. OE cells with chronic arecoline exposure resulted in increased ALDH1 population, CD44 positivity, stemness-related transcription factors (Oct4, Nanog, and Sox2), epithelial-mesenchymal transdifferentiation (EMT) traits, chemoresistance, migration/invasiveness/anchorage independent growth and in vivo tumor growth as compared to their untreated controls. Mechanistically, ectopic miR-145 over-expression in chronic arecoline-exposed OE (AOE) cells inhibited the cancer stemness and xenografic. In AOE cells, luciferase reporter assays further revealed that miR-145 directly targets the 3' UTR regions of Oct4 and Sox2 and overexpression of Sox2/Oct4 effectively reversed miR-145-regulated cancer stemness-associated phenomenas. Additionally, clinical results further revealed that Sox2 and Oct4 expression was inversely correlated with miR-145 in the tissues of areca quid chewing-associated OSCC patients. This study hence attempts to provide novel insight into areca nut-induced oral carcinogenesis and new intervention for the treatment of OSCC patients, especially in areca nut users.

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling.

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Stoyanova, Tanya; Goldstein, Andrew S; Cai, Houjian; Drake, Justin M; Huang, Jiaoti; Witte, Owen N

2012-10-15

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells

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Yélian Marc Bossou

2017-06-01

Full Text Available Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1 and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity.

[Inhibitory effect of diclofenac sodium on the proliferation of rabbit corneal epithelial cells in vitro].

Science.gov (United States)

Wu, Ningling; Du, Zhiyu

2010-11-01

To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. The fresh rabbit cornea was cultured to obtain the primary RCECs, and RCECs of passage 2 were used in this research. The cells were divided into experimental groups, the cells in which were incubated with different concentrations (18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs, and the inhibition rate was increasing along with the increasing concentration of diclofenac sodium and the incubation time(Pdiclofenac sodium, the cells in G0/G1 phase were obviously increased, and the apoptosis cusp and apoptotic rate were increased. Diclofenac sodium exerts significant inhibitory effect on RCECs in a dosage-dependent manner, and it may function by inducing cell apoptosis and ceasing cell cycles.

Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

NARCIS (Netherlands)

Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

2017-01-01

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

NARCIS (Netherlands)

Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface

Czech Academy of Sciences Publication Activity Database

Čejková, Jitka; Trošan, Peter; Čejka, Čestmír; Lencová, A.; Zajícová, Alena; Javorková, Eliška; Kubinová, Šárka; Syková, Eva; Holáň, Vladimír

2013-01-01

Roč. 116, Nov. (2013), s. 312-323 ISSN 0014-4835 R&D Projects: GA ČR GAP304/11/0653; GA ČR(CZ) GAP301/11/1568 Grant - others:GA UK(CZ) 668012; GA MŠk(CZ) SVV 265211 Institutional research plan: CEZ:AV0Z50390512 Institutional support: RVO:68378041 Keywords : corneal oxidative injury * mesenchymal stem cells * nanofiber scaffolds Subject RIV: FF - HEENT, Dentistry Impact factor: 3.017, year: 2013

Transepithelial Riboflavin Absorption in an Ex Vivo Rabbit Corneal Model.

Science.gov (United States)

Gore, Daniel M; O'Brart, David; French, Paul; Dunsby, Chris; Allan, Bruce D

2015-07-01

To measure depth-specific riboflavin concentrations in corneal stroma using two-photon fluorescence microscopy and compare commercially available transepithelial corneal collagen cross-linking (CXL) protocols. Transepithelial CXL riboflavin preparations--MedioCross TE, Ribocross TE, Paracel plus VibeX Xtra, and iontophoresis with Ricrolin+--were applied to the corneal surface of fresh postmortem rabbit eyes in accordance with manufacturers' recommendations for clinical use. Riboflavin 0.1% (VibeX Rapid) was applied after corneal epithelial debridement as a positive control. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35-μm thick were cut on a cryostat, mounted on a slide, and imaged by two-photon fluorescence microscopy. Mean (SD) concentrations were calculated from five globes tested for each protocol. Peak riboflavin concentration of 0.09% (± 0.01) was observed within the most superficial stroma (stromal depth 0-10 μm) in positive controls (epithelium-off). At the same depth, peak stromal riboflavin concentrations for MedioCross TE, Ricrolin+, Paracel/Xtra, and Ribocross TE were 0.054% (± 0.01), 0.031% (0.003), 0.021% (± 0.001), and 0.015% (± 0.004), respectively. At a depth of 300 μm (within the demarcation zone commonly seen after corneal cross-linking), the stromal concentration in epithelium-off positive controls was 0.075% (± 0.006), while at the same depth MedioCross TE and Ricrolin+ achieved 0.018% (± 0.006) and 0.016% (0.002), respectively. None of the remaining transepithelial protocols achieved concentrations above 0.005% at this same 300-μm depth. Overall, MedioCross TE was the best-performing transepithelial formulation. Corneal epithelium is a significant barrier to riboflavin absorption into the stroma. Existing commercial transepithelial CXL protocols achieve relatively low riboflavin concentrations in the anterior corneal stroma when compared to gold standard epithelium-off absorption

Long-Term Outcomes of Cultivated Limbal Epithelial Transplantation: Evaluation and Comparison of Results in Children and Adults

Directory of Open Access Journals (Sweden)

Anita Ganger

2015-01-01

Full Text Available Purpose. To compare the long-term clinical outcomes of cultivated limbal epithelial transplantation (CLET in children and adults with limbal stem cell deficiency. Design. Retrospective case series. Methods. Case records of patients with limbal stem cell deficiency (LSCD who underwent CLET from April 2004 to December 2014 were studied. Outcome measures were compared in terms of anatomical success and visual improvement. Parameters for total anatomical success were avascular, epithelized, and clinically stable corneal surface without conjunctivalization, whereas partial anatomical success was considered when mild vascularization (sparing centre of cornea and mild conjunctivalization were noted along with complete epithelization. Results. A total of 62 cases underwent the CLET procedure: 38 (61.3% were children and 24 (38.7% were adults. Patients with unilateral LSCD (33 children and 21 adults had autografts and those with bilateral LSCD (5 children and 3 adults had allografts. Amongst the 54 autografts partial and total anatomical success were noted in 21.2% and 66.6% children, respectively, and 19.0% and 80.9% in adults, respectively (p value 0.23. Visual improvement of 1 line and ≥2 lines was seen in 57.5% and 21.2% children, respectively, and 38% and 38% in adults, respectively (p value 0.31. Conclusion. Cultivated limbal epithelial transplantation gives good long-term results in patients with LSCD and the outcomes are comparable in children and adults.

Identification of putative dental epithelial stem cells in a lizard with life-long tooth replacement.

Science.gov (United States)

Handrigan, Gregory R; Leung, Kelvin J; Richman, Joy M

2010-11-01

Most dentate vertebrates, including humans, replace their teeth and yet the process is poorly understood. Here, we investigate whether dental epithelial stem cells exist in a polyphyodont species, the leopard gecko (Eublepharis macularius). Since the gecko dental epithelium lacks a histologically distinct site for stem cells analogous to the mammalian hair follicle bulge, we performed a pulse-chase experiment on juvenile geckos to identify label-retaining cells (LRCs). We detected LRCs exclusively on the lingual side of the dental lamina, which exhibits low proliferation rates and is not involved in tooth morphogenesis. Lingual LRCs were organized into pockets of high density close to the successional lamina. A subset of the LRCs expresses Lgr5 and other genes that are markers of adult stem cells in mammals. Also similar to mammalian stem cells, the LRCs appear to proliferate in response to gain of function of the canonical Wnt pathway. We suggest that the LRCs in the lingual dental lamina represent a population of stem cells, the immediate descendents of which form the successional lamina and, ultimately, the replacement teeth in the gecko. Furthermore, their location on the non-tooth-forming side of the dental lamina implies that dental stem cells are sequestered from signals that might otherwise induce them to differentiate.

Inhibiting glycogen synthase kinase-3 and transforming growth factor-β signaling to promote epithelial transition of human adipose mesenchymal stem cells.

Science.gov (United States)

Setiawan, Melina; Tan, Xiao-Wei; Goh, Tze-Wei; Hin-Fai Yam, Gary; Mehta, Jodhbir S

2017-09-02

This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor β (TGFβ) signaling. STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFβ1 receptor kinase inhibitor), A-83-01 (TGFβ type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin α5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, Epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 δNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip ® Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFβ signaling. It can be an adult stem cell source for epithelial cell-based therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Proton nuclear magnetic resonance study on the barrier function of pig corneal epithelium and endothelium

International Nuclear Information System (INIS)

Yokoi, Norihiko; Kinoshita, Shigeru; Morimoto, Taketoshi; Yoshizaki, Kazuo.

1995-01-01

Using gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) as a tracer, the barrier function of the corneal epithelium and endothelium was evaluated by proton nuclear magnetic resonance. Whole pig eyes and cornea excised with scleral rim, which had been incubated in dextran-added Gd-DTPA solution, were subjected to T 1 relaxation measurement and magnetic resonance imaging (MRI). After incubation, the T 1 relaxation rate (1/T 1 ) of the excised cornea increased to a steady value, whereas that of the cornea from the whole eye increased only slightly. These results indicated that the increase in the T 1 relaxation rate of the excised cornea was attributable to Gd-DTPA penetration from the corneal endothelium and that the corneal epithelium exhibited a strong barrier function against Gd-DTPA entry. The MRI study also confirmed the strong barrier, enhanced signals being detected within the aqueous fluid in the T 1 -weighted image only when the corneal epithelium was abraded. Since Gd-DTPA scarcely penetrates the intact corneal epithelium, Gd-DTPA-enhanced MRI shows potential as a quantitative tracer in evaluating epithelial barrier disruption. (author)

In-vivo expansion of autologous limbal stem cell using simple limbal epithelial transplantation for treatment of limbal stem cell deficiency

OpenAIRE

Lal, Ikeda; Panchal, Bhavik Uttam; Basu, Sayan; Sangwan, Virender S

2013-01-01

A 20-year-old man from Bangladesh suffered accidental alkali injury to his right eye in May 2010 leading to total limbal stem cell deficiency. An amniotic membrane graft was performed 5 days after the accident and the patient presented to our institute 6 months later. On ocular examination, his best corrected visual acuity (BCVA) was 20/50 with a 360° pannus at the periphery and central area was spared but had stromal scarring. He underwent simple limbal epithelial transplantation (SLET) taki...

Corneal Laceration

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Full Text Available ... Eye Health A-Z Symptoms Glasses & Contacts Tips & Prevention News Ask an Ophthalmologist Patient Stories Español Eye ... Causes Corneal Laceration? Corneal Laceration Diagnosis Corneal Laceration Treatment What Is Corneal Laceration? Leer en Español: ¿Qué ...

Atypical Presentation of Iridocorneal Endothelial Syndrome With Band Keratopathy but No Corneal Edema Managed With Descemet Membrane Endothelial Keratoplasty.

Science.gov (United States)

Zygoura, Vasiliki; Lavy, Itay; Verdijk, Robert M; Santander-García, Diana; Baydoun, Lamis; Dapena, Isabel; Melles, Gerrit R J

2018-04-17

To report an unusual presentation of iridocorneal endothelial (ICE) syndrome associated with band keratopathy and its management with ethylenediamine-tetraacetic acid (EDTA) chelation and Descemet membrane endothelial keratoplasty (DMEK). A 57-year-old female patient presented with unilateral progressive painless visual impairment, corneal band keratopathy, and morphological corneal endothelial changes without corneal edema or any previous ophthalmic, medical, or family history. Routine specular and confocal microscopy imaging, as well as biomicroscopy, best-corrected visual acuity, and pachymetry measurements were performed before and after the surgical procedures. Histopathologic and immunohistochemical evaluations of the surgically excised diseased DM-endothelium were performed. Superficial epithelial keratectomy with EDTA chelation was performed. After an initial period of a few months of corneal clearance, the patient presented with recurrence of visually significant band keratopathy. After 1 year, she underwent retreatment with superficial epithelial keratectomy and EDTA chelation, followed by DMEK. Histopathologic and immunohistochemical analysis showed ICE syndrome. Two years after DMEK surgery, the cornea was still clear and band keratopathy had not recurred. To the best of our knowledge, this is the first case in the literature that reports the association of ICE syndrome with band keratopathy. As band keratopathy recurred shortly after EDTA chelation, endothelial keratoplasty (DMEK) may be indicated to successfully treat such cases.

Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.

Science.gov (United States)

Hatou, Shin; Higa, Kazunari; Inagaki, Emi; Yoshida, Satoru; Kimura, Erika; Hayashi, Ryuhei; Tsujikawa, Motokazu; Tsubota, Kazuo; Nishida, Kohji; Shimmura, Shigeto

2013-12-01

Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.

Removal of the basement membrane enhances corneal wound healing.

Science.gov (United States)

Pal-Ghosh, Sonali; Pajoohesh-Ganji, Ahdeah; Tadvalkar, Gauri; Stepp, Mary Ann

2011-12-01

Recurrent corneal erosions are painful and put patients' vision at risk. Treatment typically begins with debridement of the area around the erosion site followed by more aggressive treatments. An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound created using a dulled-blade. This study was conducted to determine whether 1) inhibiting MMP9 function during healing after dulled-blade wounding impacts erosion development and 2) wounds made with a rotating-burr heal without erosions. Oral or topical inhibition of MMPs after dulled-blade wounding does not improve healing. Wounds made by rotating-burr heal with significantly fewer erosions than dulled-blade wounds. The localization of MMP9, β4 integrin and basement membrane proteins (LN332 and type VII collagen), immune cell influx, and reinnervation of the corneal nerves were compared after both wound types. Rotating-burr wounds remove the anterior basement membrane centrally but not at the periphery near the wound margin, induce more apoptosis of corneal stromal cells, and damage more stromal nerve fibers. Despite the fact that rotating-burr wounds do more damage to the cornea, fewer immune cells are recruited and significantly more wounds resolve completely. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bioactive Antimicrobial Peptides as Therapeutics for Corneal Wounds and Infections.

Science.gov (United States)

Griffith, Gina L; Kasus-Jacobi, Anne; Pereira, H Anne

2017-06-01

Significance: More than 2 million eye injuries and infections occur each year in the United States that leave civilians and military members with reduced or complete vision loss due to the lack of effective therapeutics. Severe ocular injuries and infections occur in varied settings including the home, workplace, and battlefields. In this review, we discuss the potential of developing antimicrobial peptides (AMPs) as therapeutics for the treatment of corneal wounds and infections for which the current treatment options are inadequate. Recent Advances: Standard-of-care employs the use of fluorescein dye for the diagnosis of ocular defects and is followed by the use of antibiotics and/or steroids to treat the infection and reduce inflammation. Recent advances for treating corneal wounds include the development of amniotic membrane therapies, wound chambers, and drug-loaded hydrogels. In this review, we will discuss an innovative approach using AMPs with the dual effect of promoting corneal wound healing and clearing infections. Critical Issues: An important aspect of treating ocular injuries is that treatments need to be effective and administered expeditiously. This is especially important for injuries that occur during combat and in individuals who demonstrate delayed wound healing. To overcome gaps in current treatment modalities, bioactive peptides based on naturally occurring cationic antimicrobial proteins are being investigated as new therapeutics. Future Directions: The development of new therapeutics that can treat ocular infections and promote corneal wound healing, including the healing of persistent corneal epithelial defects, would be of great clinical benefit.

Heme Oxygenase-2 as a novel target to treat inflammation and chronic neuropathic pain associated with corneal injury and surgery

OpenAIRE

Marrazzo, Giuseppina

2012-01-01

Corneal refractive surgery aims at correcting alteration of the shape of the cornea correlated with myopia, hyperopia and astigmatism. More than 12 million patients have undergone refractive surgery since it was approved (see http://www. laser-eye-surgery statistics.com/). Laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) are the most used techniques to perform experimental corneal surgery. Several studies demonstrated that after epithelial removal (first...

Silk Film Topography Directs Collective Epithelial Cell Migration

Science.gov (United States)

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

Therapeutic effects of zerumbone in an alkali-burned corneal wound healing model.

Science.gov (United States)

Kim, Jong Won; Jeong, Hyuneui; Yang, Myeon-Sik; Lim, Chae Woong; Kim, Bumseok

2017-07-01

Cornea is an avascular transparent tissue. Ocular trauma caused by a corneal alkali burn induces corneal neovascularization (CNV), inflammation, and fibrosis, leading to vision loss. The purpose of this study was to examine the effects of Zerumbone (ZER) on corneal wound healing caused by alkali burns in mice. CNV was induced by alkali-burn injury in BALB/C female mice. Topical ZER (three times per day, 3μl each time, at concentrations of 5, 15, and 30μM) was applied to treat alkali-burned mouse corneas for 14 consecutive days. Histopathologically, ZER treatment suppressed alkali burn-induced CNV and decreased corneal epithelial defects induced by alkali burns. Corneal tissue treated with ZER showed reduced mRNA levels of pro-angiogenic genes, including vascular endothelial growth factor, matrix metalloproteinase-2 and 9, and pro-fibrotic factors such as alpha smooth muscle actin and transforming growth factor-1 and 2. Immunohistochemical analysis demonstrated that the infiltration of F4/80 and/or CCR2 positive cells was significantly decreased in ZER-treated corneas. ZER markedly inhibited the mRNA and protein levels of monocyte chemoattractant protein-1 (MCP-1) in human corneal fibroblasts and murine peritoneal macrophages. Immunoblot analysis revealed that ZER decreased the activation of signal transducer and activator of transcription 3 (STAT3), with consequent reduction of MCP-1 production by these cells. In conclusion, topical administration of ZER accelerated corneal wound healing by inhibition of STAT3 and MCP-1 production. Copyright © 2017. Published by Elsevier B.V.

EMMPRIN Modulates Epithelial Barrier Function through a MMP–Mediated Occludin Cleavage

Science.gov (United States)

Huet, Eric; Vallée, Benoit; Delbé, Jean; Mourah, Samia; Prulière-Escabasse, Virginie; Tremouilleres, Magali; Kadomatsu, Kenji; Doan, Serge; Baudouin, Christophe; Menashi, Suzanne; Gabison, Eric E.

2011-01-01

Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure. PMID:21777561

Corneal Crosslinking With Rose Bengal and Green Light: Efficacy and Safety Evaluation.

Science.gov (United States)

Zhu, Hong; Alt, Clemens; Webb, Robert H; Melki, Samir; Kochevar, Irene E

2016-09-01

To evaluate crosslinking of cornea in vivo using green light activation of Rose Bengal (RGX) and assess potential damaging effects of the green light on retina and iris. Corneas of Dutch belted rabbits were de-epithelialized, then stained with Rose Bengal and exposed to green light, or not further treated. Corneal stiffness was measured by uniaxial tensiometry. Re-epithelialization was assessed by fluorescein fluorescence. Keratocytes were counted on hematoxylin and eosin (H&E)-stained sections, and iris cell damage was assessed by lactate dehydrogenase staining. Thermal effects on the blood-retinal barrier (BRB) were assessed by fluorescein angiography and those on photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris by light microscopy and transmission electron microscopy. RGX (10-min irradiation; 150 J/cm) increased corneal stiffness 1.9-fold on day 1 (1.25 ± 0.21 vs. 2.38 ± 0.59 N/mm; P = 0.036) and 2.8-fold compared with controls on day 28 (1.70 ± 0.74 vs. 4.95 ± 1.86 N/mm; P = 0.003). Keratocytes decreased only in the anterior stroma on day 1 (24.0 ± 3.0 vs. 3.67 ± 4.73, P = 0.003) and recovered by day 28 (37.7 ± 8.9 vs. 34.5 ± 2.4, P = 0.51). Iris cells were not thermally damaged. No evidence of BRB breakdown was detected on days 1 or 28. Retina from RGX-treated eyes seemed normal with RPE cells showing intact nuclei shielded apically by melanosomes, morphologically intact photoreceptor outer segments, normal outer nuclear layer thickness, and choriocapillaris containing intact erythrocytes. The substantial corneal stiffening produced by RGX together with the lack of significant effects on keratocytes and no evidence for retina or iris damage suggest that RGX-initiated corneal crosslinking may be a safe, rapid, and effective treatment.

Corneal biomechanical properties from air-puff corneal deformation imaging

Science.gov (United States)

Marcos, Susana; Kling, Sabine; Bekesi, Nandor; Dorronsoro, Carlos

2014-02-01

The combination of air-puff systems with real-time corneal imaging (i.e. Optical Coherence Tomography (OCT), or Scheimpflug) is a promising approach to assess the dynamic biomechanical properties of the corneal tissue in vivo. In this study we present an experimental system which, together with finite element modeling, allows measurements of corneal biomechanical properties from corneal deformation imaging, both ex vivo and in vivo. A spectral OCT instrument combined with an air puff from a non-contact tonometer in a non-collinear configuration was used to image the corneal deformation over full corneal cross-sections, as well as to obtain high speed measurements of the temporal deformation of the corneal apex. Quantitative analysis allows direct extraction of several deformation parameters, such as apex indentation across time, maximal indentation depth, temporal symmetry and peak distance at maximal deformation. The potential of the technique is demonstrated and compared to air-puff imaging with Scheimpflug. Measurements ex vivo were performed on 14 freshly enucleated porcine eyes and five human donor eyes. Measurements in vivo were performed on nine human eyes. Corneal deformation was studied as a function of Intraocular Pressure (IOP, 15-45 mmHg), dehydration, changes in corneal rigidity (produced by UV corneal cross-linking, CXL), and different boundary conditions (sclera, ocular muscles). Geometrical deformation parameters were used as input for inverse finite element simulation to retrieve the corneal dynamic elastic and viscoelastic parameters. Temporal and spatial deformation profiles were very sensitive to the IOP. CXL produced a significant reduction of the cornea indentation (1.41x), and a change in the temporal symmetry of the corneal deformation profile (1.65x), indicating a change in the viscoelastic properties with treatment. Combining air-puff with dynamic imaging and finite element modeling allows characterizing the corneal biomechanics in-vivo.

Corneal Laceration

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Full Text Available ... Ophthalmology/Strabismus Ocular Pathology/Oncology Oculoplastics/Orbit Refractive ... Corneal Laceration Sections What Is Corneal Laceration? Corneal Laceration Symptoms What Causes ...

Leptin and Adiponectin Modulate the Self-renewal of Normal Human Breast Epithelial Stem Cells.

Science.gov (United States)

Esper, Raymond M; Dame, Michael; McClintock, Shannon; Holt, Peter R; Dannenberg, Andrew J; Wicha, Max S; Brenner, Dean E

2015-12-01

Multiple mechanisms are likely to account for the link between obesity and increased risk of postmenopausal breast cancer. Two adipokines, leptin and adiponectin, are of particular interest due to their opposing biologic functions and associations with breast cancer risk. In the current study, we investigated the effects of leptin and adiponectin on normal breast epithelial stem cells. Levels of leptin in human adipose explant-derived conditioned media positively correlated with the size of the normal breast stem cell pool. In contrast, an inverse relationship was found for adiponectin. Moreover, a strong linear relationship was observed between the leptin/adiponectin ratio in adipose conditioned media and breast stem cell self-renewal. Consistent with these findings, exogenous leptin stimulated whereas adiponectin suppressed breast stem cell self-renewal. In addition to local in-breast effects, circulating factors, including leptin and adiponectin, may contribute to the link between obesity and breast cancer. Increased levels of leptin and reduced amounts of adiponectin were found in serum from obese compared with age-matched lean postmenopausal women. Interestingly, serum from obese women increased stem cell self-renewal by 30% compared with only 7% for lean control serum. Taken together, these data suggest a plausible explanation for the obesity-driven increase in postmenopausal breast cancer risk. Leptin and adiponectin may function as both endocrine and paracrine/juxtacrine factors to modulate the size of the normal stem cell pool. Interventions that disrupt this axis and thereby normalize breast stem cell self-renewal could reduce the risk of breast cancer. ©2015 American Association for Cancer Research.

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

Directory of Open Access Journals (Sweden)

Adrian Biddle

Full Text Available CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that

Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products.

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Doucet, O; Lanvin, M; Thillou, C; Linossier, C; Pupat, C; Merlin, B; Zastrow, L

2006-06-01

The aim of this study was to evaluate the interest of a new three-dimensional epithelial model cultivated from human corneal cells to replace animal testing in the assessment of eye tolerance. To this end, 65 formulated cosmetic products and 36 chemicals were tested by means of this in vitro model using a simplified toxicokinetic approach. The chemicals were selected from the ECETOC data bank and the EC/HO International validation study list. Very satisfactory results were obtained in terms of concordance with the Draize test data for the formulated cosmetic products. Moreover, the response of the corneal model appeared predictive of human ocular response clinically observed by ophthalmologists. The in vitro scores for the chemicals tested strongly correlated with their respective scores in vivo. For all the compounds tested, the response of the corneal model to irritants was similar regardless of their chemical structure, suggesting a good robustness of the prediction model proposed. We concluded that this new three-dimensional epithelial model, developed from human corneal cells, could be promising for the prediction of eye irritation induced by chemicals and complex formulated products, and that these two types of materials should be tested using a similar protocol. A simple shortening of the exposure period was required for the chemicals assumed to be more aggressively irritant to the epithelial tissues than the cosmetic formulae.

Correlation between epithelial thickness in normal corneas, untreated ectatic corneas, and ectatic corneas previously treated with CXL; is overall epithelial thickness a very early ectasia prognostic factor?

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Kanellopoulos, Anastasios John; Aslanides, Ioannis M; Asimellis, George

2012-01-01

To determine and correlate epithelial corneal thickness (pachymetric) measurements taken with a digital arc scanning very high frequency ultrasound biomicroscopy (HF UBM) imaging system (Artemis-II), and compare mean and central epithelial thickness among normal eyes, untreated keratoconic eyes, and keratoconic eyes previously treated with collagen crosslinking (CXL). Epithelial pachymetry measurements (topographic mapping) were conducted on 100 subjects via HF UBM. Three groups of patients were included: patients with normal eyes (controls), patients with untreated keratoconic eyes, and patients with keratoconic eyes treated with CXL. Central, mean, and peripheral corneal epithelial thickness was examined for each group, and a statistical study was conducted. Mean, central, and peripheral corneal epithelial thickness was compared between the three groups of patients. Epithelium thickness varied substantially in the keratoconic group, and in some cases there was a difference of up to 20 μm between various points of the same eye, and often a thinner epithelium coincided with a thinner cornea. However, on average, data from the keratoconic group suggested an overall thickening of the epithelium, particularly over the pupil center of the order of +3 μm, while the mean epithelium thickness was on average +1.1 μm, compared to the control population (P = 0.005). This overall thickening was more pronounced in younger patients in the keratoconic group. Keratoconic eyes previously treated with CXL showed, on average, virtually the same average epithelium thickness (mean -0.7 μm, -0.2 μm over the pupil center, -0.9 μm over the peripheral zone) as the control group. This finding further reinforces our novel theory of the "reactive" component of epithelial thickening in corneas that are biomechanically unstable, becoming stable when biomechanical rigidity is accomplished despite persistence of cornea topographic irregularity. A highly irregular epithelium may be

Epithelial ingrowth under a laser in situ keratomileusis flap after phacoemulsification.

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Braunstein, Richard E; Airiani, Suzanna; Chang, Stanley

2003-11-01

A 47-year-old man was referred to us for management of a cataract in the left eye. The patient had an ocular history of high myopia with anisometropia, amblyopia in the left eye, and stable myopic lattice degeneration in both eyes. The patient had successful bilateral laser in situ keratomileusis 3 years before and multiple retinal surgeries for treatment of a rhegmatogenous retinal detachment associated with a giant retinal tear in the temporal region of the retina with subsequent proliferative vitreoretinopathy. Phacoemulsification was performed uneventfully. A single interrupted 10-0 nylon suture was placed in the temporal clear corneal wound and removed 7 weeks postoperatively. One month later, slitlamp examination revealed a 1.5 mm tongue-like area of epithelial ingrowth under the corneal flap. The epithelial cells seemed to enter the flap-stroma interface through the previously placed suture tract and advanced centrally.

Self-Assembled Matrix by Umbilical Cord Stem Cells

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Biagio Saitta

2011-09-01

Full Text Available Corneal integrity is critical for vision. Corneal wounds frequently heal with scarring that impairs vision. Recently, human umbilical cord mesenchymal stem cells (cord stem cells have been investigated for tissue engineering and therapy due to their availability and differentiation potential. In this study, we used cord stem cells in a 3-dimensional (3D stroma-like model to observe extracellular matrix organization, with human corneal fibroblasts acting as a control. For 4 weeks, the cells were stimulated with a stable Vitamin C (VitC derivative ±TGF-b1. After 4 weeks, the mean thickness of the constructs was ~30 mm; however, cord stem cell constructs had 50% less cells per unit volume, indicating the formation of a dense matrix. We found minimal change in decorin and lumican mRNA, and a significant increase in perlecan mRNA in the presence of TGF-b1. Keratocan on the other hand decreased with TGF-b1 in both cell lineages. With both cell types, the constructs possessed aligned collagen fibrils and associated glycosaminoglycans. Fibril diameters did not change with TGF-b1 stimulation or cell lineage; however, highly sulfated glycosaminoglycans associated with the collagen fibrils significantly increased with TGF-b1. Overall, we have shown that cord stem cells can secrete their own extracellular matrix and promote the deposition and sulfation of various proteoglycans. Furthermore, these cells are at least comparable to commonly used corneal fibroblasts and present an alternative for the 3D in vitro tissue engineered model.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.

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Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-ichiro

2013-12-01

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Tectonic DSAEK for the Management of Impending Corneal Perforation

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Enrique O. Graue-Hernandez

2012-01-01

Full Text Available Purpose. To report a case of severe corneal thinning secondary to dry eye treated with a tectonic Descemet stripping automated lamellar keratoplasty (DSAEK and amniotic membrane graft. Methods. A 72-year-old man with a history of long standing diabetes mellitus type 2 and dry eye presented with 80% corneal thinning and edema on the right eye and no signs of infectious disease, initially managed with topical unpreserved lubrication and 20% autologous serum drops. Eight weeks after, the defect advanced in size and depth until Descemetocele was formed. Thereafter, he underwent DSAEK for tectonic purposes. One month after the procedure, the posterior lamellar graft was well adhered but a 4 mm epithelial defect was still present. A multilayered amniotic membrane graft was then performed. Results. Ocular surface healed quickly and reepithelization occurred over a 2-week period. Eight months after, the ocular surface remained stable and structurally adequate. Conclusion. Tectonic DSAEK in conjunction with multilayered amniotic graft may not only provide structural support and avoid corneal perforation, but may also promote reepithelization and ocular surface healing and decrease concomitant inflammation.

Engineering of Corneal Tissue through an Aligned PVA/Collagen Composite Nanofibrous Electrospun Scaffold.

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Wu, Zhengjie; Kong, Bin; Liu, Rui; Sun, Wei; Mi, Shengli

2018-02-24

Corneal diseases are the main reason of vision loss globally. Constructing a corneal equivalent which has a similar strength and transparency with the native cornea, seems to be a feasible way to solve the shortage of donated cornea. Electrospun collagen scaffolds are often fabricated and used as a tissue-engineered cornea, but the main drawback of poor mechanical properties make it unable to meet the requirement for surgery suture, which limits its clinical applications to a large extent. Aligned polyvinyl acetate (PVA)/collagen (PVA-COL) scaffolds were electrospun by mixing collagen and PVA to reinforce the mechanical strength of the collagen electrospun scaffold. Human keratocytes (HKs) and human corneal epithelial cells (HCECs) inoculated on aligned and random PVA-COL electrospun scaffolds adhered and proliferated well, and the aligned nanofibers induced orderly HK growth, indicating that the designed PVA-COL composite nanofibrous electrospun scaffold is suitable for application in tissue-engineered cornea.

Role of corneal collagen fibrils in corneal disorders and related pathological conditions

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Hong-Yan Zhou

2017-05-01

Full Text Available The cornea is a soft tissue located at the front of the eye with the principal function of transmitting and refracting light rays to precisely sense visual information. Corneal shape, refraction, and stromal stiffness are to a large part determined by corneal fibrils, the arrangements of which define the corneal cells and their functional behaviour. However, the modality and alignment of native corneal collagen lamellae are altered in various corneal pathological states such as infection, injury, keratoconus, corneal scar formation, and keratoprosthesis. Furthermore, corneal recuperation after corneal pathological change is dependent on the balance of corneal collagen degradation and contraction. A thorough understanding of the characteristics of corneal collagen is thus necessary to develop viable therapies using the outcome of strategies using engineered corneas. In this review, we discuss the composition and distribution of corneal collagens as well as their degradation and contraction, and address the current status of corneal tissue engineering and the progress of corneal cross-linking.

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

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Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

2016-03-10

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

Effectiveness of Photodynamic Therapy in the Healing of Corneal Alkali Burn in Rats

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Akshay Khera

2016-06-01

Full Text Available In this study, we investigated the effect of photodynamic therapy (PDT on the healing of corneal alkali burns in rats. The experiment was performed on 50 adult non-linear rats. Depending on the intervention, the animals were divided into 5 equal groups with 10 animals in each group: Group 1 included rats with intact eyes (the control group and Groups 2 through 5 were experimental groups with EAB. Group 2 consisted of rats subjected to instillation of 0.25% chloramphenicol solution; Group 3 consisted of rats subjected to photodynamic irradiation according to our scheme: 300 mJ (630 nm for 3 minutes; Group 4 consisted of rats subjected to instillation of methylene blue (MB; Group 5 consisted of rats subjected to instillation of MB with subsequent photodynamic irradiation according to the described scheme. During all periods of observation, the infiltration of the subcorneal zone was less pronounced in Group 5 than in the other groups and was represented mainly by round cells in the anterior chamber, iris, retina, and ciliary zone. The instillation of MB with subsequent photodynamic irradiation was the most effective in reducing the bacterial contamination Thus, PDT with the photosensitizer methylene blue, in accordance with the designed exposure mode, provided the epithelialization and bacteriostatic effect during corneal repair after EAB. In conclusion, PDT improves a wound’s healing process, which is expressed in the reduction of inflammatory infiltration and the promotion of corneal epithelialization.

Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects.

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Pauloin, Thierry; Dutot, Mélody; Liang, Hong; Chavinier, Emilie; Warnet, Jean-Michel; Rat, Patrice

2009-10-01

The aim of this study was to investigate high-molecular-weight hyaluronan (HA-HMW) corneal protection against sodium lauryl sulfate (SLS)-induced toxic effects with in vitro and in vivo experimental approaches. In vitro experiments consisted of a human corneal epithelial cell line incubated with HA-HMW, rinsed, and incubated with SLS. Cell viability, oxidative stress, chromatin condensation, caspase-3, -8, -9, and P2X7 cell death receptor activation, interleukin-6, and interleukin-8 production were investigated. In vivo experiments consisted of 36 New Zealand white rabbits treated for 3 days, 3 times per day, with HA-HMW or phosphate-buffered salt solution. At day 4, eyes were treated with SLS. Clinical observation and in vivo confocal microscopy using the Rostock Cornea Module of the Heidelberg Retina Tomograph-II were performed to evaluate and to compare SLS-induced toxicity between eyes treated with HA-HMW and eyes treated with phosphate-buffered salt solution. In vitro data indicate that exposure of human corneal epithelial cells to HA-HMW significantly decreased SLS-induced oxidative stress, apoptosis, and inflammation cytokine production. In vivo data indicate that SLS cornea injuries, characterized by damaged corneal epithelium, damaged anterior stroma, and inflammatory infiltrations, were attenuated with HA-HMW treatment. A good correlation was seen between in vitro and in vivo findings showing that HA-HMW decreases SLS-induced toxic effects and protects cornea.

Stabilization of collagen nanofibers with l-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells

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Lai JY

2014-11-01

provide a useful strategy to assist in the development of carbodiimide cross-linked amniotic membrane as a stable stem cell niche for corneal epithelial tissue engineering.Keywords: amniotic membrane, L-lysine-pretreated concentration, collagen nanofiber stabilization, limbal progenitor cell preservation, corneal epithelial tissue engineering

The non-contact ("air puff") tonometer: variability and corneal staining.

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Myers, K J; Scott, C A

1975-01-01

We investigated the possibility of significant corneal trauma (as revealed by slit lamp observation of the fluorescein instilled eye), and massage effects following determination of intraocular pressure with the A. O. Non-Contact tonometer (NCT). Fifteen different, normal human eyes were each applanated 150 successive times with the NCT; leading to the conclusion that only minor, superficial corneal epithelial defects sometimes resulted and that, in line with other studies, the initially higher readings (about 1 mm), obtained with the NCT, were most likely due to patient apprehension, while the subsequently lower readings represented patient acceptance of the process and were not a result of true aqueous massage. As in an earlier study, we found the instrument's variability to be about plus and minus 1 or plus and minus 2 mm and probably due to the subject's own cardiac cycle.

Effect of heme oxygenase-1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro.

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Cao, Yi; Wu, Ben-Juan; Zheng, Wei-Ping; Yin, Ming-Li; Liu, Tao; Song, Hong-Li

2017-07-01

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase-1 (HO-1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO-1 recombinant adenovirus (HO-MSCs) for stable expression of HO-1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor-α (TNF-α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad-MSCs, Ad-HO + MSCs or HO-MSCs. mRNA and protein expression of Zona occludens-1 (ZO-1) and human HO-1 and the release of cytokines were measured. ZO-1 and human HO-1 in Caco2 were significantly decreased after treatment with TNF-α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO-1 was not significantly affected by Caco2 treatment with TNF-α, Ad-HO, and MSCs. In contrast, ZO-1 and human HO-1 increased significantly when the damaged Caco2 was treated with HO-MSCs. HO-MSCs showed the strongest effect on the expression of ZO-1 in colon epithelial cells. Coculture with HO-MSCs showed the most significant effects on reducing the expression of IL-2, IL-6, IFN-γ and increasing the expression of IL-10. HO-MSCs protected the intestinal epithelial barrier, in which endogenous HO-1 was involved. HO-MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti-inflammatory factors. These results suggested that HO-MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO-1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

The effect of excimer laser keratectomy on corneal glutathione-related enzymes in rabbits.

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Bilgihan, Ayşe; Bilgihan, Kamil; Yis, Ozgür; Yis, Nilgün Safak; Hasanreisoglu, Berati

2003-04-01

Glutathione related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Excimer laser is a very useful tool for the treatment of refractive errors and removing superficial corneal opacities. Previous studies have shown that excimer laser may initiate free radical formation in the cornea. In the present study, we evaluated the effect of excimer laser keratectomy on corneal glutathione-related enzyme activities in rabbits. Animals were divided into five groups, and all groups were compared with the controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy (PRK) (group 3), traditional PRK (group 4) and deep traditional PRK (group 5). Corneal glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) activities were measured after 24h. Corneal GPx and GR activities significantly decreased only in group 5 (p < 0.05) but GST activities significantly decreased in all groups when compared with the control group (p < 0.05). In conclusion, excimer laser inhibits the glutathione dependent defense system in the cornea, this effect becomes more prominent after high doses of excimer laser energy and antioxidants may be useful to reduce free radical mediated complications.

Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

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Shu-Long Wang

2015-01-01

Full Text Available AIM: To investigate into the potential involvement of pyrin containing 3 gene (NLRP3, a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses. METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1 (HSV-1. Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40 (SV40-immortalized human corneal epithelial cell line were also examined. Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β. RESULTS: The NLRP3 activation induced by HSV-1 infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore, in the SV40-immortalized human corneal epithelial cells, NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium (known as an inhibitor of NLRP3 activation effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot. CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

Evaluation of Topical Cyclosporine in Preventing the Development of Corneal Haze after Photorefractive Keratectomy

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2014-05-13

NAME OF RESPONSIBLE PERSON: a. REPORT UNCLASSIFIED b. ABSTRACT UNCLASSIFIED c. THIS PAGE UNCLASSIFIED 19b. TELEPHONE NUMBER...fibroblast proliferation and concomitant fibrotic scar deposition at the corneal wound site. Unfortunately, mitomycin C also inhibits epithelial...stromal, and endothelial replication and can lead to vascular endothelial injury and secondary tissue necrosis. 3 Due to these side effects, steroids are

The preservative polyquaternium-1 increases cytoxicity and NF-kappaB linked inflammation in human corneal epithelial cells

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Paimela, Tuomas; Ryhänen, Tuomas; Kauppinen, Anu; Marttila, Liisa; Salminen, Antero

2012-01-01

Purpose In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell culture. Methods HCE-2 cells were exposed to the commercial eye drops Travatan, Systane Ultra, Xalatan, and the preservative BAK. Cell viability was determined using colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by release of lactate dehydrogenase (LDH). Induction of apoptosis was measured with a using a colorimetric caspase-3 assay kit. DNA binding of the nuclear factor kappa B (NF-κB) transcription factor, and productions of the proinflammatory cytokines, interleukins IL-6 and IL-8, were determined using an enzyme-linked immunosorbent assay (ELISA) method. Results Cell viability, as measured by the MTT assay, declined by up to 50% after exposure to Travatan or Systane Ultra solutions which contain 0.001% PQ-1. BAK at 0.02% rather than at 0.001% concentration evoked total cell death signs on HCE-2 cells. In addition, cell membrane permeability, as measured by LDH release, was elevated by sixfold with Travatan and by a maximum threefold with Systane Ultra. Interestingly, Travatan and Systane Ultra activated NF-κB and elevated the secretion of inflammation markers IL-6 by 3 to eightfold and IL-8 by 1.5 to 3.5 fold, respectively, as analyzed with ELISA. Conclusions Eye drops containing PQ-1 evoke cytotoxicity and enhance the NF-κB driven inflammation reaction in cultured HCE-2 cells. Our results indicate that these harmful effects of ocular solutions preserved with PQ-1 should be further evaluated in vitro and in vivo. PMID:22605930

The effect of excimer laser keratectomy on corneal glutathione peroxidase activities and aqueous humor selenium levels in rabbits.

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Yis, Ozgür; Bilgihan, Ayşe; Bilgihan, Kamil; Yis, Nilgün Safak; Hasanreisoğlu, Berati

2002-06-01

The formation of free oxygen radicals has been demonstrated in the corneal tissue after 193 nm laser irradiation. Cornea has several defense mechanisms that protect against oxidative damage. One of them, glutathione peroxidase (GPx), catalyzes the destruction of hydrogen peroxide and lipid hydroperoxide. Selenium is a trace element which is incorporated into the selenoenzyme GPx. In the present study, the effect of excimer laser keratectomy on corneal GPx activities and aqueous humor selenium concentrations in rabbits was evaluated. Animals were divided into five groups, and all groups were compared: controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy(PRK; group 3), superficial traditional PRK (50 microm; group 4) and deep traditional PRK (100 microm; group 5). Corneal GPx activities were measured by a modification of the coupled assay procedure. Aqueous humor selenium concentrations were determined using hydride generation atomic absorption spectrometry. Corneal GPx activities were significantly lower only in group 5 ( P<0.05), and the selenium concentration in the aqueous humor did not change in any group. Deep corneal photoablation inhibits GPx enzyme activities in the cornea. Therefore, antioxidants may be useful in reducing free radical-mediated complications after excimer laser corneal photoablation.

Unilateral congenital corneal keloid with anterior segment mesenchymal dysgenesis and subluxated lens: case report and review of literature.

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Vanathi, M; Sen, Seema; Panda, Anita; Dada, Tanuj; Behera, Geeta; Khokhar, Sudharshan

2007-01-01

To report the unusual association of unilateral congenital corneal keloid with anterior-segment mesenchymal dysgenesis and bilateral subluxated lens. A 20-year old man presented with a mass lesion involving the left cornea. The corneal lesion had been present since birth. On biomicroscopic examination, a well-defined vascularized, grayish-white mass occupying the whole of the left cornea was seen. The right eye showed multiple peripheral corneal opacities with iridocorneal adhesions, a poorly defined supranasal limbus, and a subluxated lens. Excision biopsy of the mass was done for histopathologic examination. Histopathologic examination of the excised corneal mass showed features consistent with that of a corneal keloid: thickened keratinized epithelium, absent Bowman membrane layer, and fibrovascular hyperplasia composed of hyalinized collagen fibers with irregular orientation of the collagen lamellae. During penetrating keratoplasty of the left eye, an anomalous iris pattern with poorly defined angle and a supranasal subluxated lens was also observed. Extraction of the subluxated lens was also done. The graft failed subsequent to a nonhealing persistent epithelial defect. Our case report highlights the rare association of a unilateral congenital corneal keloid with anterior-segment mesenchymal dysgenesis and bilateral subluxated lens.

Corneal densitometry and its correlation with age, pachymetry, corneal curvature, and refraction.

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Garzón, Nuria; Poyales, Francisco; Illarramendi, Igor; Mendicute, Javier; Jáñez, Óscar; Caro, Pedro; López, Alfredo; Argüeso, Francisco

2017-12-01

To determine normative corneal densitometry values in relation to age, sex, refractive error, corneal thickness, and keratometry, measured using the Oculus Pentacam system. Three hundred and thirty-eight healthy subjects (185 men; 153 women) with no corneal disease underwent an exhaustive ocular examination. Corneal densitometry was expressed in standardized grayscale units (GSU). The mean corneal densitometry over the total area was 16.46 ± 1.85 GSU. The Pearson correlation coefficient for total densitometry was r = 0.542 (p  0.05). This is the first report of normative corneal densitometry values in relation to keratometry, corneal thickness, and spherical equivalent measured with the latest Oculus Pentacam software. Corneal densitometry increases with age, but corneal keratometry and refractive parameters do not affect light scattering in the human cornea.

Intracellular Position of Centrioles and the Direction of Homeostatic Epithelial Cell Movements in the Mouse Cornea.

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Silverman, Erika; Zhao, Jin; Merriam, John C; Nagasaki, Takayuki

2017-02-01

Corneal epithelial cells exhibit continuous centripetal movements at a rate of about 30 µm per day, but neither the driving force nor the mechanism that determines the direction of movements is known. To facilitate the investigation of homeostatic cell movement, we examined if the intracellular position of a centriole can be used as a directional marker of epithelial cell movements in the mouse cornea. A direction of cell movements was estimated in fixed specimens from a pattern of underlying subepithelial nerve fibers. Intracellular position of centrioles was determined by gamma-tubulin immunohistology and plotted in a narrow strip along the entire diameter of a cornea from limbus to limbus. When we determined the position of centrioles in the peripheral cornea where cell movements proceed generally along a radial path, about 55% of basal epithelial cells contained a centriole in the front half of a cell. However, in the central cornea where cells exhibit a spiral pattern of movements, centrioles were distributed randomly. These results suggest that centrioles tend to be positioned toward the direction of movement in corneal basal epithelial cells when they are moving centripetally at a steady rate.

Related research on corneal higher-order aberrations after different ways refractive surgery

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Shu-Xi He

2015-08-01

Full Text Available AIM:To evaluate the changes of corneal high-order aberration(including Coma, Spab, RMShafter laser in situ keratomileusis(LASIKwith femtosecond laser, sub-Bowman keratomileusis(SBKand laser epithelial keratomileusis(LASEK.METHODS: Of 82 myopic patients(164 eyes, 31 patients(62 eyeswere treated by FS-LASIK, 31 patients(62 eyeswere treated by SBK, 20 patients(40 eyeswere treated by LASEK. Sirius system was used for measuring the coma aberration, spherical aberration, and high order aberration at 1, 15d,1, 3mo after surgery.RESULTS: 1Vision: The uncorrected visual acuity of the three groups had no differences(P>0.05. 2Corneal aberrations: Three kinds of surgical procedure for patients with corneal aberration had significant impact. The C7, C8, C12 and RMSh of three groups were increased significantly(P0.05. The C7, C8, C12 and RMSh were not recovered to preoperative levels after 3mo. But the increase of patients after FS-LASIK was smaller than the other two groups, with statistical significance(P0.05.CONCLUSION: Compared with SBK and LASEK,FS-LASIK has better visual acuity in the early postoperative and corneal higher-order aberrations increase is relatively small.

The corneal epithelium: clinical relevance of cytokine-mediated responses to maintenance of corneal health O epitélio da córnea: relevância clínica das respostas mediadas por citocinas para manter a saúde da córnea

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PS Reinach

2008-12-01

Full Text Available We review the growth factor receptor-mediated cell signaling events that induce the responses required for the maintenance of corneal epithelial health. Our focus is to show how such responses contribute to sustaining corneal transparency and deturgescence, so basic to the pathogenesis of corneal diseases. Furthermore, we point out how alterations of receptor-mediated control of these responses account for losses in corneal transparency. In particular, the roles of growth factors in the mediation of normal corneal function, including epithelial cell proliferation, prevention of compromise of the barrier function of the cornea, and maintenance of normal renewal processes are discussed in relation to clinical entities involving the cornea.Revimos os eventos de sinalização celular mediados por receptores de fatores de crescimento, usados para manter a saúde do epitélio da córnea. O objetivo é mostrar como essas respostas contribuem para manter a transparência e a deturgescência da córnea, críticos na patogênese das doenças da córnea. Mais ainda, enfatizamos como alterações no controle mediado por receptor dessas respostas contribuem na transparência da córnea. Especificamente, o papel dos fatores de crescimento na mediação do controle funcional normal da córnea, incluindo proliferação epitelial, prevenção da quebra da função de barreira, manutenção do processo de renovação são discutidos em relação às entidades clínicas envolvidas na córnea.

Hevin plays a pivotal role in corneal wound healing.

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Shyam S Chaurasia

Full Text Available BACKGROUND: Hevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK in hevin-null (hevin(-/- mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin to rescue the stromal cellular components damaged by the excimer laser. METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT and hevin (-/- mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM, transmission electron microscopy (TEM, immunohistochemistry (IHC and western blotting (WB. IHC showed upregulation of hevin in IrrPTK-injured WT mice. Hevin (-/- mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts, inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice. CONCLUSIONS/SIGNIFICANCE: Hevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. Hevin (-/- mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued

Pharmacological activities of an eye drop containing Matricaria chamomilla and Euphrasia officinalis extracts in UVB-induced oxidative stress and inflammation of human corneal cells.

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Bigagli, Elisabetta; Cinci, Lorenzo; D'Ambrosio, Mario; Luceri, Cristina

2017-08-01

Ultraviolet B (UVB) exposure is a risk factor for corneal damage resulting in oxidative stress, inflammation and cell death. The aim of this study was to investigate the potential protective effects of a commercial eye drop (Dacriovis™) containing Matricaria chamomilla and Euphrasia officinalis extracts on human corneal epithelial cells (HCEC-12) against UVB radiation-induced oxidative stress and inflammation as well as the underlying mechanisms. The antioxidant potential of the eye drops was evaluated by measuring the ferric reducing antioxidant power and the total phenolic content by Folin-Ciocalteu reagent. HCEC-12 cells were exposed to UVB radiation and treated with the eye drops at various concentrations. Cell viability, wound healing assay, reactive oxygen species (ROS) levels, protein and lipid oxidative damage and COX-2, IL-1β, iNOS, SOD-2, HO-1 and GSS gene expression, were assessed. Eye drops were able to protect corneal epithelial cells from UVB-induced cell death and ameliorated the wound healing; the eye drops exhibited a strong antioxidant activity, decreasing ROS levels and protein and lipid oxidative damage. Eye drops also exerted anti-inflammatory activities by decreasing COX-2, IL-1β, iNOS expression, counteracted UVB-induced GSS and SOD-2 expression and restored HO-1 expression to control levels. These findings suggest that an eye drop containing Matricaria chamomilla and Euphrasia officinalis extracts exerts positive effects against UVB induced oxidative stress and inflammation and may be useful in protecting corneal epithelial cells from UVB exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Acyclovir-Loaded Albumin Nanoparticles and Improvement of Acyclovir Permeation Across Human Corneal Epithelial T Cells.

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Suwannoi, Panita; Chomnawang, Mullika; Sarisuta, Narong; Reichl, Stephan; Müller-Goymann, Christel C

2017-12-01

The aim of the present study was to develop acyclovir (ACV) ocular drug delivery systems of bovine serum albumin (BSA) nanoparticles as well as to assess their in vitro transcorneal permeation across human corneal epithelial (HCE-T) cell multilayers. The ACV-loaded BSA nanoparticles were prepared by desolvation method along with physicochemical characterization, cytotoxicity, as well as in vitro transcorneal permeation studies across HCE-T cell multilayers. The nanoparticles appeared to be spherical in shape and nearly uniform in size of about 200 nm. The size of nanoparticles became smaller with decreasing BSA concentration, while the ratios of water to ethanol seemed not to affect the size. Increasing the amount of ethanol in desolvation process led to significant reduction of drug entrapment of nanoparticles with smaller size and more uniformity. The ACV-loaded BSA nanoparticles prepared were shown to have no cytotoxic effect on HCE-T cells used in permeation studies. The in vitro transcorneal permeation results revealed that ACV could permeate through the HCE-T cell multilayers significantly higher from BSA nanoparticles than from aqueous ACV solutions. The ACV-loaded BSA nanoparticles could be prepared by desolvation method without glutaraldehyde in the formulation. ACV could increasingly permeate through the multilayers of HCE-T cells from the ACV-loaded BSA nanoparticles. Therefore, the ACV-loaded BSA nanoparticles could be a highly potential ocular drug delivery system.

Kinetics of corneal epithelium turnover in vivo. Studies of lovastatin

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Cenedella, R.J.; Fleschner, C.R.

1990-01-01

The authors developed a direct chemical approach for estimating the rate of turnover of the corneal epithelium in vivo. The method was used to examine the effects of lovastatin, a potent inhibitor of cholesterol biosynthesis, on proliferation and turnover of the epithelium. Corneal DNA was labeled by pulse injection (IP) of the rat with 3H-thymidine, and 3H-labeled DNA was recovered from peripheral and central corneas over the next 15 days. Only the epithelium became labeled, and the loss of label by cell desquamation began 3 days after injection. The loss of 3H-DNA from the cornea (peripheral plus central region) followed first-order kinetics. The half-life of the disappearance was about 3 days. The peripheral cornea became more highly labeled than the central cornea and began to lose 3H-DNA before the central cornea. These observations support the possibility of a higher mitotic rate in the peripheral region and the centripetal movement of a population of peripheral epithelial cells in the normal cornea. The half-lives of the disappearance of 3H-DNA from peripheral and central corneas measured between days 5 and 15 postinjection were identical, both at 3 days. Complete turnover of the corneal epithelium would, therefore, require about 2 weeks (4-5 half-lives). Treatment of the rat with lovastatin had no obvious effects upon the proliferation or turnover of the corneal epithelium. Although lovastatin inhibited corneal 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol synthesis, the cornea compensated by induction of this enzyme so that there was no net inhibition of cholesterol synthesis in the cornea

Cultivated Oral Mucosa Epithelium in Ocular Surface Reconstruction in Aniridia Patients

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Dariusz Dobrowolski

2015-01-01

Full Text Available Purpose. Efficacy of cultivated oral mucosa epithelial transplantation (COMET procedure in corneal epithelium restoration of aniridia patients. Methods. Study subjects were aniridia patients (13 patients; 17 eyes with irregular, vascular conjunctival pannus involving visual axis who underwent autologous transplantation of cultivated epithelium. For the procedure oral mucosa epithelial cells were obtained from buccal mucosa with further enzymatic treatment. Suspension of single cells was seeded on previously prepared denuded amniotic membrane. Cultures were carried on culture dishes inserts in the presence of the inactivated with Mitomycin C monolayer of 3T3 fibroblasts. Cultures were carried for seven days. Stratified oral mucosa epithelium with its amniotic membrane carrier was transplanted on the surgically denuded corneal surface of aniridia patients with total or subtotal limbal stem cell deficiency. Outcome Measures. Corneal surface, epithelial regularity, and visual acuity improvement were evaluated. Results. At the end of the observation period, 76.4% of the eyes had regular transparent epithelium and 23.5% had developed epithelial defects or central corneal haze; in 88.2% of cases visual acuity had increased. VA range was from HM 0.05 before the surgery to HM up to 0.1 after surgery. Conclusion. Application of cultivated oral mucosa epithelium restores regular epithelium on the corneal surface with moderate improvement in quality of vision.

Progress of research on corneal collagen cross-linking for corneal melting

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Ke-Ren Xiao

2016-06-01

Full Text Available Corneal collagen cross-linking(CXLcould increase the mechanical strength, biological stability and halt ectasia progression due to covalent bond formed by photochemical reaction between ultraviolet-A and emulsion of riboflavin between collagen fibers in corneal stroma. Corneal melting is an autoimmune related noninfectious corneal ulcer. The mechanism of corneal melting, major treatment, the basic fundamental of ultraviolet-A riboflavin induced CXL and the clinical researches status and experiment in CXL were summarized in the study.

[Effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats].

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Zhao, B; Wu, G F; Zhang, Y J; Zhang, W; Yang, F F; Xiao, D; Zeng, K X; Shi, J H; Su, L L; Hu, D H

2017-01-20

Objective: To investigate the effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats. Methods: (1) Human amniotic epithelial stem cells were isolated from the amnion tissue of 5 full-term pregnant women in Department of Obstetrics of our hospital by the method of trypsin digestion, and their morphology was observed. The third passage of cells were stained with rhodamine-phalloidin for cytoskeleton observation. The third passage of cells were identified with flow cytometry through the detection of expressions of cell surface markers CD29, CD31, CD34, CD90, CD105, SSEA3, SSEA4 and immunity-related marker human leukocyte antigen-D related site (HLA-DR). The third passage of cells were also assessed the ability of adipogenic and osteogenic differentiation. (2) The third passage of human amniotic epithelial stem cells were cultured in DMEM medium supplemented with 10% exosome-free fetal bovine serum. Exosomes were isolated from culture supernatant by the method of ultracentrifugation and represented with scanning electron microscope for morphologic observation. (3) Six adult SD rats were anesthetized, and four 1 cm×1 cm sized wounds with full-thickness skin defect were made on the back of each rat. The wounds on the back of each rat were divided into control group, 25 μg/mL exosomes group, 50 μg/mL exosomes group, and 100 μg/mL exosomes group according to the random number table (with 6 wounds in each group), and a total volume of 100 μL phosphate buffered saline, 25 μg/mL exosomes, 50 μg/mL exosomes, and 100 μg/mL exosomes were evenly injected around the wound through multiple subcutaneous sites, respectively. The wound healing rate was calculated based on measurement on post injury day (PID) 7, 14, and 21. On PID 21, the healed wound tissue of each group was collected and stained with HE to observe and count skin accessories, and the arrangement of collagen fibers was observed with Masson

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

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Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

2014-11-28

Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

International Nuclear Information System (INIS)

Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

2014-01-01

Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

Polycomb Repressive Complex 2 Enacts Wnt Signaling in Intestinal Homeostasis and Contributes to the Instigation of Stemness in Diseases Entailing Epithelial Hyperplasia or Neoplasia.

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Oittinen, Mikko; Popp, Alina; Kurppa, Kalle; Lindfors, Katri; Mäki, Markku; Kaikkonen, Minna U; Viiri, Keijo

2017-02-01

Canonical Wnt/β-catenin signaling regulates the homeostasis of intestinal epithelium by controlling the balance between intestinal stem cell self-renewal and differentiation but epigenetic mechanisms enacting the process are not known. We hypothesized that epigenetic regulator, Polycomb Repressive Complex-2 (PRC2), is involved in Wnt-mediated epithelial homeostasis on the crypt-villus axis and aberrancies therein are implicated both in celiac disease and in intestinal malignancies. We found that PRC2 establishes repressive crypt and villus specific trimethylation of histone H3 lysine 27 (H3K27me3) signature on genes responsible for, for example, nutrient transport and cell killing in crypts and, for example, proliferation and differentiation in mature villi, suggesting that PRC2 facilitates the Wnt-governed intestinal homeostasis. When celiac patients are on gluten-containing diet PRC2 is out-of-bounds active and consequently its target genes were found affected in intestinal epithelium. Significant set of effective intestinal PRC2 targets are also differentially expressed in colorectal adenoma and carcinomas. Our results suggest that PRC2 gives rise and maintains polar crypt and villus specific H3K27me3 signatures. As H3K27me3 is a mark enriched in developmentally important genes, identified intestinal PRC2 targets are possibly imperative drivers for enterocyte differentiation and intestinal stem cell maintenance downstream to Wnt-signaling. Our work also elucidates the mechanism sustaining the crypt hyperplasia in celiac disease and suggest that PRC2-dependent fostering of epithelial stemness is a common attribute in intestinal diseases in which epithelial hyperplasia or neoplasia prevails. Finally, this work demonstrates that in intestine PRC2 represses genes having both pro-stemness and pro-differentiation functions, fact need to be considered when designing epigenetic therapies including PRC2 as a drug target. Stem Cells 2017;35:445-457. © 2016 Alpha

Corneal edema and keratitis following selective laser trabeculoplasty.

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Liu, Erica Tan; Seery, Loren S; Arosemena, Analisa; Lamba, Tania; Chaya, Craig J

2017-06-01

To describe three cases of keratitis following Selective Laser Trabeculoplasty (SLT). Three females with a history of glaucoma presented with corneal edema, keratitis (endothelial, epithelial) and decreased visual acuity shortly after SLT. There was variable resolution of symptoms after starting treatment with oral antiherpetics and topical steroids. With the increase in usage of SLT as a treatment for glaucoma and subsequent reports of keratitis, it is imperative for ophthalmic surgeons to be aware of herpes simplex as a possible risk factor. Prompt treatment with antivirals and steroids can potentially prevent scarring and permanent damage to the cornea.

Efficacy of iontophoresis-assisted epithelium-on corneal cross-linking for keratoconus

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Hong-Zhen Jia

2018-04-01

Full Text Available Corneal cross-linking (CXL is a noninvasive therapeutic procedure for keratoconus that is aimed at improving corneal biomechanical properties by induction of covalent cross-links between stromal proteins. It is accomplished by ultraviolet A (UVA radiation of the cornea, which is first saturated with photosensitizing riboflavin. It has been shown that standard epithelium-off CXL (S-CXL is efficacious, and it has been recommended as the standard of care procedure for keratoconus. However, epithelial removal leads to pain, transient vision loss, and a higher risk of corneal infection. To avoid these disadvantages, transepithelial CXL was developed. Recently, iontophoresis has been adopted to increase riboflavin penetration through the epithelium. Several clinical observations have demonstrated the safety and efficacy of iontophoresis-assisted epithelium-on CXL (I-CXL for keratoconus. This review aimed to provide a comprehensive summary of the published studies regarding I-CXL and a comparison between I-CXL and S-CXL. All articles used in this review were mainly retrieved from the PubMed database. Original articles and reviews were selected if they were related to the I-CXL technique or related to the comparison between I-CXL and S-CXL.

Intraocular pressure, corneal thickness, and corneal hysteresis in Steinert's myotonic dystrophy

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Carlos Alexandre de A. Garcia Filho

2011-06-01

Full Text Available PURPOSE: Low intraocular pressure (IOP measured by Goldmann applanation tonometry (GAT is one of the ocular manifestations of Steinert's myotonic dystrophy. The goal of this study was to evaluate the corneal-compensated IOP as well as corneal properties (central corneal thickness and corneal hysteresis in patients with myotonic dystrophy. METHODS: A total of 12 eyes of 6 patients with Steinert's myotonic dystrophy (dystrophy group and 12 eyes of 6 age-, race-, and gender-matched healthy volunteers (control group were included in the study. GAT, Dynamic Contour Tonometry (DCT-Pascal and Ocular Response Analyzer (ORA were used to assess the IOP. Central corneal thickness was obtained by ultrasound pachymetry, and corneal hysteresis was analyzed using the ORA device. In light of the multiplicity of tests performed, the significance level was set at 0.01 rather than 0.05. RESULTS: The mean (standard deviation [SD] GAT, DCT, and corneal-compensated ORA IOP in the dystrophy group were 5.4 (1.4 mmHg, 9.7 (1.5 mmHg, and 10.1 (2.6 mmHg, respectively. The mean (SD GAT, DCT, and corneal-compensated ORA IOP in the control group was 12.6 (2.9 mmHg, 15.5 (2.7 mmHg, and 15.8 (3.4 mmHg, respectively. There were significant differences in IOP values between dystrophy and control groups obtained by GAT (mean, -7.2 mmHg; 99% confidence interval [CI], -10.5 to -3.9 mmHg; P<0.001, DCT (mean, -5.9 mmHg; 99% CI, -8.9 to -2.8 mmHg; P<0.001, and corneal-compensated ORA measurements (mean, -5.7 mmHg; 99% CI, -10.4 to -1.0 mmHg; P=0.003. The mean (SD central corneal thickness was similar in the dystrophy (542 [31] µm and control (537 [11] µm groups (P=0.65. The mean (SD corneal hysteresis in the dystrophy and control groups were 11.2 (1.5 mmHg and 9.7 (1.2 mmHg, respectively (P=0.04. CONCLUSIONS: Patients with Steinert's myotonic dystrophy showed lower Goldmann and corneal-compensated IOP in comparison with healthy individuals. Since central corneal thickness and

New Details of the Human Corneal Limbus Revealed With Second Harmonic Generation Imaging.

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Park, Choul Yong; Lee, Jimmy K; Zhang, Cheng; Chuck, Roy S

2015-09-01

To report novel findings of the human corneal limbus by using second harmonic generation (SHG) imaging. Corneal limbus was imaged by using an inverted two-photon excitation fluorescence microscope. Laser (Ti:Sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of SHG and autofluorescence (AF) were collected through a 425/30-nm emission filter and a 525/45-emission filter, respectively. Multiple, consecutive, and overlapping image stacks (z-stack) were acquired for the corneal limbal area. Two novel collagen structures were revealed by SHG imaging at the limbus: an anterior limbal cribriform layer and presumed anchoring fibers. Anterior limbal cribriform layer is an intertwined reticular collagen architecture just beneath the limbal epithelial niche and is located between the peripheral cornea and Tenon's/scleral tissue. Autofluorescence imaging revealed high vascularity in this structure. Central to the anterior limbal cribriform layer, radial strands of collagen were found to connect the peripheral cornea to the limbus. These presumed anchoring fibers have both collagen and elastin and were found more extensively in the superficial layers than deep layer and were absent in very deep limbus near Schlemm's canal. By using SHG imaging, new details of the collagen architecture of human corneal limbal area were elucidated. High resolution images with volumetric analysis revealed two novel collagen structures.

Intrastromal corneal ring implants for corneal thinning disorders: an evidence-based analysis.

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2009-01-01

The purpose of this project was to determine the role of corneal implants in the management of corneal thinning disease conditions. An evidence-based review was conducted to determine the safety, effectiveness and durability of corneal implants for the management of corneal thinning disorders. The evolving directions of research in this area were also reviewed. SUBJECT OF THE EVIDENCE-BASED ANALYSIS: The primary treatment objectives for corneal implants are to normalize corneal surface topography, improve contact lens tolerability, and restore visual acuity in order to delay or defer the need for corneal transplant. Implant placement is a minimally invasive procedure that is purported to be safe and effective. The procedure is also claimed to be adjustable, reversible, and both eyes can be treated at the same time. Further, implants do not limit the performance of subsequent surgical approaches or interfere with corneal transplant. The evidence for these claims is the focus of this review. The specific research questions for the evidence review were as follows: SafetyCorneal Surface Topographic Effects:Effects on corneal surface remodellingImpact of these changes on subsequent interventions, particularly corneal transplantation (penetrating keratoplasty [PKP])Visual AcuityRefractive OutcomesVISUAL QUALITY (SYMPTOMS): such as contrast vision or decreased visual symptoms (halos, fluctuating vision)Contact lens toleranceFunctional visual rehabilitation and quality of lifePatient satisfaction:Disease Process:Impact on corneal thinning processEffect on delaying or deferring the need for corneal transplantation TARGET POPULATION AND CONDITION Corneal ectasia (thinning) comprises a range of disorders involving either primary disease conditions such as keratoconus and pellucid marginal corneal degeneration or secondary iatrogenic conditions such as corneal thinning occurring after LASIK refractive surgery. The condition occurs when the normally round dome-shaped cornea

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

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Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K

2018-02-01

The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils

A Comparison of Different Corneal Iontophoresis Protocols for Promoting Transepithelial Riboflavin Penetration.

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Gore, Daniel M; O'Brart, David P; French, Paul; Dunsby, Chris; Allan, Bruce D

2015-12-01

To measure corneal riboflavin penetration using different transepithelial iontophoresis protocols. Freshly enucleated rabbit eyes were divided into nine treatment groups of 4 eyes. One group, in which 0.1% wt/vol riboflavin was applied for 30 minutes without iontophoresis after corneal epithelial debridement, acted as a control. The remaining groups were treated with an intact epithelium using different riboflavin formulations and varying iontophoresis current, soak, and rinse times. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35 μm thick were then imaged immediately by two-photon fluorescence microscopy, using image processing software to quantify stromal riboflavin concentration at different corneal depths. In the epithelium-on iontophoresis treatment groups, greater stromal riboflavin penetration was achieved with higher-concentration riboflavin solutions, greater iontophoresis dosage, and longer solution contact times. A protocol utilizing 0.25% wt/vol riboflavin with benzalkonium chloride (BAC) 0.01% and two cycles of applied current and subsequent soaking (1 mA 5 minutes, soak 5 minutes; 0.5 mA 5 minutes, soak 5 minutes) achieved similar stromal riboflavin penetration to epithelium-off controls. The best-performing non-BAC-containing protocol produced stromal riboflavin penetration approximately 60% that of epithelium-off controls. Riboflavin solutions containing saline resulted in minimal stromal penetration. Riboflavin loading within the epithelium was equivalent to or higher than that in the subjacent stroma, despite rinsing the ocular surface with balanced salt solution. Modified iontophoresis protocols can significantly improve transepithelial riboflavin penetration in experimental corneal collagen cross-linking.

TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

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Martínez-Rendón, Jacqueline; Sánchez-Guzmán, Erika; Rueda, Angélica; González, James; Gulias-Cañizo, Rosario; Aquino-Jarquín, Guillermo; Castro-Muñozledo, Federico; García-Villegas, Refugio

2017-07-01

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca 2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Confocal comparison of corneal reinnervation after small incision lenticule extraction (SMILE) and femtosecond laser in situ keratomileusis (FS-LASIK).

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Li, Meiyan; Niu, Lingling; Qin, Bing; Zhou, Zimei; Ni, Katherine; Le, Qihua; Xiang, Jun; Wei, Anji; Ma, Weiping; Zhou, Xingtao

2013-01-01

To evaluate corneal reinnervation, and the corresponding corneal sensitivity and keratocyte density after small incision lenticule extraction (SMILE) and femtosecond laser in situ keratomileusis (FS-LASIK). In this prospective, non-randomized observational study, 18 patients (32 eyes) received SMILE surgery, and 22 patients (42 eyes) received FS-LASIK surgery to correct myopia. The corneal subbasal nerve density and microscopic morphological changes in corneal architecture were evaluated by confocal microscopy prior to surgery and at 1 week, 1 month, 3 months, and 6 months after surgery. A correlation analysis was performed between subbasal corneal nerve density and the corresponding keratocyte density and corneal sensitivity. The decrease in subbasal nerve density was less severe in SMILE-treated eyes than in FS-LASIK-treated eyes at 1 week (P = 0.0147), 1 month (P = 0.0243), and 3 months (P = 0.0498), but no difference was detected at the 6-month visit (P = 0.5277). The subbasal nerve density correlated positively with central corneal sensitivity in both groups (r = 0.416, PLASIK group, respectively). The SMILE-treated eyes have a lower risk of developing peripheral empty space with epithelial cells filling in (P = 0.0005). The decrease in subbasal nerve fiber density was less severe in the SMILE group than the FS-LASIK group in the first 3 months following the surgeries. The subbasal nerve density was correlated with central corneal sensitivity.

Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges.

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Purba, Talveen S; Haslam, Iain S; Poblet, Enrique; Jiménez, Francisco; Gandarillas, Alberto; Izeta, Ander; Paus, Ralf

2014-05-01

Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology. © 2014 WILEY Periodicals, Inc.

Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human Filamin A (FLNA diseases

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Douvaras Panagiotis

2012-02-01

Full Text Available Abstract Background Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human FLNA/+ females, heterozygous for X-linked, filamin A gene (FLNA mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. FlnaDilp2/+ mice, heterozygous for an X-linked filamin A (Flna nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of FlnaDilp2/+ mice was affected in any way that might predict abnormal corneal epithelial maintenance. Results X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver of FlnaDilp2/+ and wild-type (WT female X-inactivation mosaics, hemizygous for the X-linked, LacZ reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of FlnaDilp2/+ and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in FlnaDilp2/+ corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually, consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in FlnaDilp2/+ compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of FlnaDilp2/+ than wild-type Flna+/+ X-inactivation mosaics. Conclusions Mosaic analysis identified no major effect of the mouse FlnaDilp2 mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.

Tissue and cellular biomechanics during corneal wound injury and repair.

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Raghunathan, Vijay Krishna; Thomasy, Sara M; Strøm, Peter; Yañez-Soto, Bernardo; Garland, Shaun P; Sermeno, Jasmyne; Reilly, Christopher M; Murphy, Christopher J

2017-08-01

Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical cues, as well as cellular responses to the intrinsic chemistry and biophysical attributes associated with the matrix of the wound space. Here, we document how the biomechanics of the corneal stroma are altered through the course of wound repair following keratoablative procedures in rabbits. Further we documented the influence that substrate stiffness has on stromal cell mechanics. Following corneal epithelial debridement, New Zealand white rabbits underwent phototherapeutic keratectomy (PTK) on the right eye (OD). Wound healing was monitored using advanced imaging modalities. Rabbits were euthanized and corneas were harvested at various time points following PTK. Tissues were characterized for biomechanics with atomic force microscopy and with histology to assess inflammation and fibrosis. Factor analysis was performed to determine any discernable patterns in wound healing parameters. The matrix associated with the wound space was stiffest at 7days post PTK. The greatest number of inflammatory cells were observed 3days after wounding. The highest number of myofibroblasts and the greatest degree of fibrosis occurred 21days after wounding. While all clinical parameters returned to normal values 400days after wounding, the elastic modulus remained greater than pre-surgical values. Factor analysis demonstrated dynamic remodeling of stroma occurs between days 10 and 42 during corneal stromal wound repair. Elastic modulus of the anterior corneal stroma is dramatically altered following PTK and its changes coincide initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Factor analysis demonstrates strongest correlation between elastic modulus, myofibroblasts, fibrosis and stromal haze thickness, and between edema and central corneal

Omega-3 supplementation is neuroprotective to corneal nerves in dry eye disease: a pilot study.

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Chinnery, Holly R; Naranjo Golborne, Cecilia; Downie, Laura E

2017-07-01

To investigate whether oral, long-chain omega-3 (ω-3) essential fatty acid (EFA) supplementation, for 3 months, induces changes to the central corneal sub-basal nerve plexus in dry eye disease and whether nerve alterations correlate with clinical findings. This prospective, comparative study involved the final 12 participants enrolled in a randomised, double-masked, placebo-controlled clinical trial of 60 participants with moderate dry eye disease. Participants received either placebo (olive oil 1500 mg/day; n = 4) or ω-3 EFA supplements (~1000 mg/day eicosapentaenoic acid + ~500 mg/day docosahexaenoic acid; n = 8) for 90 days. The main outcome measure was the mean change in central corneal sub-basal plexus nerve parameters between days one and 90, quantified using in vivo confocal microscopy. Secondary outcomes included mean change in tear osmolarity, corneal dendritic cell density and basal epithelial cell density. Compared with baseline, the reduction in OSDI score and tear osmolarity at day 90 were greater in the ω-3 EFA group than the placebo group (OSDI: ω-3 EFA, mean ± SEM: -15.6 ± 2.8 vs placebo: -2.8 ± 4.1 units, t 5 = 2.6, p = 0.04; tearosmolarity: ω-3 EFA: -22.63 ± 5.7 vs placebo: -8 ± 2.7 mOsmol/L, t 9 = 2.3, p = 0.04). At day 90, corneal total nerve branch density (CTBD: 91.1 ± 8.6 vs 45.1 ± 13.4 branches/mm 2 , F 1,10 = 14, p = 0.004) and corneal nerve branch density on the main fibre (CNBD: 63.4 ± 6.5 vs 27.9 ± 11.5 branches/mm 2 , F 1,10 = 6, p = 0.03) were higher in the ω-3 EFA group compared with placebo. Relative to day 1, CNBD (branches/mm 2 ) increased at day 90 in the ω-3 EFA group (+20.0 ± 9.2, t 8 = 3.2 p = 0.01) compared with placebo (-10.8 ± 3.2). Similar changes were evident for corneal nerve fibre length (CNFL, mm/mm 2 ), which increased from baseline at day 90 in the omega-3 EFA group (+2.9 ± 1.6, t 8 = 3.4 p = 0.01) compared with placebo (-2.7 ± 0.5). There was a negative correlation between CTBD and tear osmolarity

Snail1 induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics

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Dang, Hien; Ding, Wei; Emerson, Dow; Rountree, C Bart

2011-01-01

Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT. Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation. TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation

Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

DEFF Research Database (Denmark)

Fanelli, Giorgia; Gonzalez-Cordero, Anai; Gardner, Peter J

2017-01-01

induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression......, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions....

Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation.

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Thomasy, Sara M; Raghunathan, Vijay Krishna; Miyagi, Hidetaka; Evashenk, Alexander T; Sermeno, Jasmyne C; Tripp, Geneva K; Morgan, Joshua T; Murphy, Christopher J

2018-05-01

The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or

Kinetic analysis of the rate of corneal wound healing in Otsuka long-evans Tokushima Fatty rats, a model of type 2 diabetes mellitus.

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Nagai, Noriaki; Murao, Takatoshi; Okamoto, Norio; Ito, Yoshimasa

2010-01-01

Diabetic keratopathy is a well-known ocular complication secondary to type 2 diabetes mellitus. In this study, we performed a kinetic analysis of corneal wound healing in Long-Evans rats (normal rat) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus. Corneal wound healing in 7-week-old normal rats was mostly complete 24 h after corneal epithelial abrasion, and the process of corneal wound healing took place according to an equation with a first-order rate constant. The rate of corneal wound healing in normal rats decreased with aging. The process of corneal wound healing in 38- and 60-week-old normal and OLETF rats occurred in two phases with rate constants for the first and second phases represented as alpha and beta, respectively. The alpha and beta values in 38- and 60-week-old OLETF rats were lower than those in normal rats of the corresponding age. Furthermore, a close relationship was observed between the corneal wound healing rate constant and plasma glucose levels in OLETF rats. The present studies suggest the sequence of events that occur following damage to the corneal surface in OLETF rats as a model animal for a human type 2 diabetes mellitus.

Donor-derived stem-cells and epithelial mesenchymal transition in squamous cell carcinoma in transplant recipients.

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Verneuil, Laurence; Leboeuf, Christophe; Bousquet, Guilhem; Brugiere, Charlotte; Elbouchtaoui, Morad; Plassa, Louis-François; Peraldi, Marie-Noelle; Lebbé, Celeste; Ratajczak, Philippe; Janin, Anne

2015-12-08

Skin squamous-cell-carcinoma (SCC), is the main complication in long-term kidney-transplant recipients, and it can include donor-derived cells. Preclinical models demonstrated the involvement of epithelial mesenchymal transition (EMT) in the progression of skin SCC, and the role of Snail, an EMT transcription factor, in cancer stem-cell survival and expansion.Here, we studied stem-cells and EMT expression in SCCs and concomitant actinic keratoses (AK) in kidney-transplant recipients. In SCC and AK in 3 female recipients of male kidney-transplants, donor-derived Y chromosome in epidermal stem cells was assessed using combined XY-FISH/CD133 immunostaining, and digital-droplet-PCR on laser-microdissected CD133 expressing epidermal cells.For EMT study, double immunostainings of CD133 with vimentin or snail and slug, electron microscopy and immunostainings of keratinocytes junctions were performed. Digital droplet PCR was used to check CDH1 (E-cadherin) expression level in laser-microdissected cells co-expressing CD133 and vimentin or snail and slug.The numbers of Y-chromosome were assessed using digital droplet PCR in laser-microdissected cells co-expressing CD133 and vimentin, or snail and slug, and in CD133 positive cells not expressing any EMT maker. We identified donor-derived stem-cells in basal layers and invasive areas in all skin SCCs and in concomitant AKs, but not in surrounding normal skin.The donor-derived stem-cells expressed the EMT markers, vimentin, snail and slug in SCCs but not in AKs. The expression of the EMT transcription factor, SNAI1, was higher in stem-cells when they expressed vimentin. They were located in invasive areas of SCCs. In these areas, the expressions of claudin-1 and desmoglein 1 were reduced or absent, and within the basal layer there were features of basal membrane disappearance.Donor-derived stem cells were in larger numbers in stem cells co-expressing vimentin or snail and slug than in stem cells not expressing any EMT marker

The relationship between corneal biomechanical properties and confocal microscopy findings in normal and keratoconic eyes.

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Hurmeric, Volkan; Sahin, Afsun; Ozge, Gokhan; Bayer, Atilla

2010-06-01

To investigate the relationship between corneal biomechanical properties and confocal microscopy (CM) findings in normal and keratoconic eyes. The study consisted of 28 eyes of 28 healthy volunteers and 23 eyes of 15 patients with keratoconus. The diagnosis of keratoconus was made with corneal topography and clinical findings. The corneal hysteresis (CH) and corneal resistance factor (CRF) were measured by the ocular response analyzer. In vivo CM was performed with NIDEK Confoscan 3. CH and CRF were compared with corneal morphological findings (detailed cell counts of endothelial, stromal, and epithelial cells) in vivo. CH was 10.1 +/- 1.3 mm Hg in normal eyes and 7.4 +/- 1.5 mm Hg in keratoconic eyes (P < 0.0001). CRF was 10.1 +/- 1.8 mm Hg in normal eyes and 6.2 +/- 1.4 mm Hg in keratoconic eyes (P < 0.0001). CH and CRF were negatively correlated with full-thickness stromal keratocyte density (P < 0.01; r = -0.52 and P < 0.001; r = -0.67, respectively) in healthy eyes. Keratocyte density of the posterior half of the stroma was found to be significantly related with CRF in healthy eyes (beta = -0.404; P = 0.01). There was no significant relationship among CH, CRF, and CM findings in eyes with keratoconus. There is a significant relationship between CRF and keratocyte density of the posterior half of the stroma in healthy eyes. Our results suggest that corneal elasticity is related to not only stromal matrix but also cellular structure of the cornea.

Equine corneal stromal abscesses

DEFF Research Database (Denmark)

Henriksen, M. D. L.; Andersen, P. H.; Plummer, C. E.

2013-01-01

The last 30 years have seen many changes in the understanding of the pathogenesis and treatment of equine corneal stromal abscesses (SAs). Stromal abscesses were previously considered an eye problem related to corneal bacterial infection, equine recurrent uveitis, corneal microtrauma and corneal....... Medical and surgical treatments are now directed towards elimination of fungal and bacterial infections, reduction and replacement of diseased corneal stroma, and suppression of iridocyclitis. If the abscess and anterior uveitis do not respond satisfactorily to medical therapy, full thickness or split...

Bilateral Epithelial Defects after Laser in situ Keratomileusis. Clinical Features, Management and Outcome

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Rao Srinivas

2005-03-01

Full Text Available PURPOSE: To describe the preoperative characteristics, intraoperative details, management, and postoperative in patients with bilateral epithelial defects after laser in situ keratomileusis (LASIK. METHODS: Retrospective non-comparative case series. RESULTS: Six patients with bilateral epithelial defects after LAISK were part of a cohort of 605 patients undergoing bilateral LASIK at our center from December 2001 to April 2003. The mean age of the patients (5M:1F was 28.5 7.9 years, and the average pretreatment myopic spherical equivalent (SE refraction was 7.3 0.7 D (-4, -12.25D. An epithelial flap was present in 6 eyes and an epithelial defect with a mean diameter of 3 mm (2mm, 6mm was seen in 6 eyes. In four patients the epithelial disturbance was bilaterally similar. All defects occurred in the inferior cornea and the epithelial flaps had the hinge positioned superiorly. None of the patients had ocular or systemic risk factors that could have resulted in this complication. A bandage contact lens was used in 6 eyes. At last follow-up of 5.5 9.5 months (0.25, 21 months, unaided visual acuity was 6/9 or better in 10 eyes. Best spectacle-corrected visual acuity (BSCVA was maintained in 8 eyes, while 4 eyes lost one line of BSCVA. Recurrent corneal erosions were not reported in the follow-up period. CONCLUSIONS: These patients represent a hitherto unrecognised group of individuals who appear to have a subclinical weakness of adhesion of the corneal epithelium to the underlying structures, which is not evident on clinical examination. This results in bilateral epithelial disturbances after LASIK. Appropriate management results in satisfactory clinical outcomes. Other options for treatment of the fellow eye of such patients include the use of a different microkeratome, release of suction during the reverse pass of the Hansatome microkeratome, and photorefractive keratectomy if the refractive error is low.

Polyoxyethylene hydrogenated castor oil modulates benzalkonium chloride toxicity: comparison of acute corneal barrier dysfunction induced by travoprost Z and travoprost.

Science.gov (United States)

Uematsu, Masafumi; Kumagami, Takeshi; Shimoda, Kenichiro; Kusano, Mao; Teshima, Mugen; To, Hideto; Kitahara, Takashi; Kitaoka, Takashi; Sasaki, Hitoshi

2011-10-01

To determine the element that modulates benzalkonium chloride (BAC) toxicity by using a new electrophysiological method to evaluate acute corneal barrier dysfunction induced by travoprost Z with sofZia (Travatan Z(®)), travoprost with 0.015% BAC (Travatan(®)), and its additives. Corneal transepithelial electrical resistance (TER) was measured in live white Japanese rabbits by 2 Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea. We evaluated corneal TER changes after a 60-s exposure to travoprost Z, travoprost, and 0.015% BAC. Similarly, TER changes were evaluated after corneas were exposed for 60 s to the travoprost additives ethylenediaminetetraacetic acid disodium salt, boric acid, mannitol, trometamol, and polyoxyethylene hydrogenated castor oil 40 (HCO-40) with or without BAC. Corneal damage was examined after exposure to BAC with or without travoprost additives using scanning electron microscopy (SEM) and a cytotoxicity assay. Although no decreases of TER were noted after exposure to travoprost Z with sofZia and travoprost with 0.015% BAC, a significant decrease of corneal TER was observed after 0.015% BAC exposure. With the exception of BAC, no corneal TER decreases were observed for any travoprost additives. After corneal exposure to travoprost additives with BAC, HCO-40 was able to prevent the BAC-induced TER decrease. SEM observations and the cytotoxicity assay confirmed that there was a remarkable improvement of BAC-induced corneal epithelial toxicity after addition of HCO-40 to the BAC. Travoprost Z with sofZia and travoprost with BAC do not induce acute corneal barrier dysfunction. HCO-40 provides protection against BAC-induced corneal toxicity.

Dynamic corneal deformation response and integrated corneal tomography

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Marcella Q Salomão

2018-01-01

Full Text Available Measuring corneal biomechanical properties is still challenging. There are several clinical applications for biomechanical measurements, including the detection of mild or early forms of ectatic corneal diseases. This article reviews clinical applications for biomechanical measurements provided by the Corvis ST dynamic non contact tonometer

Confocal comparison of corneal reinnervation after small incision lenticule extraction (SMILE and femtosecond laser in situ keratomileusis (FS-LASIK.

Directory of Open Access Journals (Sweden)

Meiyan Li

Full Text Available PURPOSE: To evaluate corneal reinnervation, and the corresponding corneal sensitivity and keratocyte density after small incision lenticule extraction (SMILE and femtosecond laser in situ keratomileusis (FS-LASIK. METHODS: In this prospective, non-randomized observational study, 18 patients (32 eyes received SMILE surgery, and 22 patients (42 eyes received FS-LASIK surgery to correct myopia. The corneal subbasal nerve density and microscopic morphological changes in corneal architecture were evaluated by confocal microscopy prior to surgery and at 1 week, 1 month, 3 months, and 6 months after surgery. A correlation analysis was performed between subbasal corneal nerve density and the corresponding keratocyte density and corneal sensitivity. RESULTS: The decrease in subbasal nerve density was less severe in SMILE-treated eyes than in FS-LASIK-treated eyes at 1 week (P = 0.0147, 1 month (P = 0.0243, and 3 months (P = 0.0498, but no difference was detected at the 6-month visit (P = 0.5277. The subbasal nerve density correlated positively with central corneal sensitivity in both groups (r = 0.416, P<0.0001, and r = 0.2567, P = 0.0038 for SMILE group and FS-LASIK group, respectively. The SMILE-treated eyes have a lower risk of developing peripheral empty space with epithelial cells filling in (P = 0.0005. CONCLUSIONS: The decrease in subbasal nerve fiber density was less severe in the SMILE group than the FS-LASIK group in the first 3 months following the surgeries. The subbasal nerve density was correlated with central corneal sensitivity.

Enhancement of cancer stem-like and epithelial−mesenchymal transdifferentiation property in oral epithelial cells with long-term nicotine exposure: Reversal by targeting SNAIL

International Nuclear Information System (INIS)

Yu, Cheng-Chia; Chang, Yu-Chao

2013-01-01

Cigarette smoking is one of the major risk factors in the development and further progression of tumorigenesis, including oral squamous cell carcinoma (OSCC). Recent studies suggest that interplay cancer stem-like cells (CSCs) and epithelial−mesenchymal transdifferentiation (EMT) properties are responsible for the tumor maintenance and metastasis in OSCC. The aim of the present study was to investigate the effects of long-term exposure with nicotine, a major component in cigarette, on CSCs and EMT characteristics. The possible reversal regulators were further explored in nicotine-induced CSCs and EMT properties in human oral epithelial (OE) cells. Long-term exposure with nicotine was demonstrated to up-regulate ALDH1 population in normal gingival and primary OSCC OE cells dose-dependently. Moreover, long-term nicotine treatment was found to enhance the self-renewal sphere-forming ability and stemness gene signatures expression and EMT regulators in OE cells. The migration/cell invasiveness/anchorage independent growth and in vivo tumor growth by nude mice xenotransplantation assay was enhanced in long-term nicotine-stimulated OE cells. Knockdown of Snail in long-term nicotine-treated OE cells was found to reduce their CSCs properties. Therapeutic delivery of Si-Snail significantly blocked the xenograft tumorigenesis of long-term nicotine-treated OSCC cells and largely significantly improved the recipient survival. The present study demonstrated that the enrichment of CSCs coupled EMT property in oral epithelial cells induced by nicotine is critical for the development of OSCC tumorigenesis. Targeting Snail might offer a new strategy for the treatment of OSCC patients with smoking habit. -- Highlights: ► Sustained nicotine treatment induced CSCs properties of oral epithelial cells. ► Long-term nicotine treatment enhance EMT properties of oral epithelial cells. ► Long-term nicotine exposure increased tumorigenicity of oral epithelial cells. ► Si

Enhancement of cancer stem-like and epithelial−mesenchymal transdifferentiation property in oral epithelial cells with long-term nicotine exposure: Reversal by targeting SNAIL

Energy Technology Data Exchange (ETDEWEB)

Yu, Cheng-Chia [Institute of Oral Science, Chung Shan Medical University, Taichung, Taiwan (China); School of Dentistry, Chung Shan Medical University, Taichung, Taiwan (China); Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Chang, Yu-Chao, E-mail: cyc@csmu.edu.tw [School of Dentistry, Chung Shan Medical University, Taichung, Taiwan (China); Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan (China)

2013-02-01

Cigarette smoking is one of the major risk factors in the development and further progression of tumorigenesis, including oral squamous cell carcinoma (OSCC). Recent studies suggest that interplay cancer stem-like cells (CSCs) and epithelial−mesenchymal transdifferentiation (EMT) properties are responsible for the tumor maintenance and metastasis in OSCC. The aim of the present study was to investigate the effects of long-term exposure with nicotine, a major component in cigarette, on CSCs and EMT characteristics. The possible reversal regulators were further explored in nicotine-induced CSCs and EMT properties in human oral epithelial (OE) cells. Long-term exposure with nicotine was demonstrated to up-regulate ALDH1 population in normal gingival and primary OSCC OE cells dose-dependently. Moreover, long-term nicotine treatment was found to enhance the self-renewal sphere-forming ability and stemness gene signatures expression and EMT regulators in OE cells. The migration/cell invasiveness/anchorage independent growth and in vivo tumor growth by nude mice xenotransplantation assay was enhanced in long-term nicotine-stimulated OE cells. Knockdown of Snail in long-term nicotine-treated OE cells was found to reduce their CSCs properties. Therapeutic delivery of Si-Snail significantly blocked the xenograft tumorigenesis of long-term nicotine-treated OSCC cells and largely significantly improved the recipient survival. The present study demonstrated that the enrichment of CSCs coupled EMT property in oral epithelial cells induced by nicotine is critical for the development of OSCC tumorigenesis. Targeting Snail might offer a new strategy for the treatment of OSCC patients with smoking habit. -- Highlights: ► Sustained nicotine treatment induced CSCs properties of oral epithelial cells. ► Long-term nicotine treatment enhance EMT properties of oral epithelial cells. ► Long-term nicotine exposure increased tumorigenicity of oral epithelial cells. ► Si

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

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Bottos, Katia M.; Oliveira, Anselmo G.; Bersanetti, Patrícia A.; Nogueira, Regina F.; Lima-Filho, Acácio A. S.; Cardillo, José A.; Schor, Paulo; Chamon, Wallace

2013-01-01

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion. PMID:23785497

Ocular dimensions, corneal thickness, and corneal curvature in quarter horses with hereditary equine regional dermal asthenia.

Science.gov (United States)

Badial, Peres R; Cisneros-Àlvarez, Luis Emiliano; Brandão, Cláudia Valéria S; Ranzani, José Joaquim T; Tomaz, Mayana A R V; Machado, Vania M; Borges, Alexandre S

2015-09-01

The aim of this study was to compare ocular dimensions, corneal curvature, and corneal thickness between horses affected with hereditary equine regional dermal asthenia (HERDA) and unaffected horses. Five HERDA-affected quarter horses and five healthy control quarter horses were used. Schirmer's tear test, tonometry, and corneal diameter measurements were performed in both eyes of all horses prior to ophthalmologic examinations. Ultrasonic pachymetry was performed to measure the central, temporal, nasal, dorsal, and ventral corneal thicknesses in all horses. B-mode ultrasound scanning was performed on both eyes of each horse to determine the dimensions of the ocular structures and to calculate the corneal curvature. Each corneal region examined in this study was thinner in the affected group compared with the healthy control group. However, significant differences in corneal thickness were only observed for the central and dorsal regions. HERDA-affected horses exhibited significant increases in corneal curvature and corneal diameter compared with unaffected animals. The ophthalmologic examinations revealed mild corneal opacity in one eye of one affected horse and in both eyes of three affected horses. No significant between-group differences were observed for Schirmer's tear test, intraocular pressure, or ocular dimensions. Hereditary equine regional dermal asthenia-affected horses exhibit decreased corneal thickness in several regions of the cornea, increased corneal curvature, increased corneal diameter, and mild corneal opacity. Additional research is required to determine whether the increased corneal curvature significantly impacts the visual accuracy of horses with HERDA. © 2014 American College of Veterinary Ophthalmologists.

A case of unilateral circumscribed posterior keratoconus evaluated by three different imaging tools: optical coherence tomography, videokeratography, and Scheimpflug corneal tomography.

Science.gov (United States)

Spadea, Leopoldo; Maraone, Giorgia; Cagini, Carlo

2017-02-01

Posterior keratoconus is a rare corneal anomaly which is part of the ectatic corneal disorders. We report a clinical presentation of a unilateral posterior keratoconus in a 42-year-old man. At the time of presentation, corrected distance visual acuity (CDVA) was 20/20 with a correction of +2.50 +2.50 × 90° in the right eye and 20/40 with +1 +3.00 × 105° in the left eye. Slit lamp microscopy showed in the left eye an evidence of corneal thinning with a mild anterior protrusion and a remarkable posterior excavation. The intraocular pressure was 19 mmHg in right eye and 16 mmHg in left eye. Ultrasound pachymetry showed a minimum corneal thickness of 556 μ in right eye and 289 μ in left eye. The anterior segment optical coherence tomography (AS-OCT) revealed central corneal thinning and showed a reduced epithelial thickness. Videokeratography showed an increase of the corneal curvature in a defined area with central steepening in the area of the posterior corneal depression with gradual paracentral flattening. The description of this case underlines the importance of this instruments such us AS-OCT and corneal topography in diagnosis of posterior keratoconus. It can also be observed that in the contralateral eye there are no signs of ectasia as in the rare condition of unilateral keratoconus.

Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

Energy Technology Data Exchange (ETDEWEB)

Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing, E-mail: caijingmmm@hotmail.com; Wang, Zehua, E-mail: zehuawang@163.net

2015-09-10

Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

International Nuclear Information System (INIS)

Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing; Wang, Zehua

2015-01-01

Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs