Kizhakkethara, I; Li, X; el-Sayed, S; Khoobehi, B; Moshfeghi, D M; Rahimy, M; Peyman, G A
Daunorubicin is a cytotoxic drug, which, in nontoxic doses, is effective in preventing cellular proliferation in experimental vitreoretinopathy. We studied dose and clearance of daunorubicin in various ocular tissues using fluorophotometry techniques. In vitro tests: The emission of fluorescence from the daunorubicin solution having a concentration range of 0.1 to 10 micrograms/mL in phosphate buffer was measured using an excitation wavelength range of 489 +/- 10 nm. The emission of fluorescence was measured at 514 nm; the linearity of the response was determined using linear regression analysis. There is a fluorescence peak of daunorubicin at 485 nm. The validity and reproducibility of the method were examined. In vivo tests: The rabbits were randomized into three groups and daunorubicin concentrations of 4, 6, or 8 micrograms/mL were injected into the vitreous. Fluorophotometry scanning from the retina to the anterior chamber was performed with a commercially available fluorophotometer at various times up to 48 hours after injection to quantify fluorescence emission of daunorubicin. The standard curve of fluorescence versus concentration of daunorubicin was linear in the range of 0.1 to 8 micrograms/mL. It was sensitive up to 0.1 microgram. The daunorubicin time concentration profile showed a dose response relationship over the 48-hour period studied. The half-life of daunorubicin in the vitreous was about 5 hours. We performed fluorophotometry using a fluorophotometer whose exciter emits light at 489 nm, which is very close to an absorption peak of daunorubicin. These two values are close enough to obviate the need for modifying the commercial fluorophotometer. Therefore the concentration of daunorubicin in the vitreous cavity can be measured noninvasively.
Dickmann, Leslie J; Yip, Victor; Li, Cuiwen; Abundes, Juan; Maia, Mauricio; Young, Cynthia; Stainton, Shannon; Hass, Philip E; Joseph, Sean B; Prabhu, Saileta; Boswell, C Andrew
In this work, we assessed the ability of fluorophotometry to measure the vitreal pharmacokinetics (PK) of fluorescently-labeled ranibizumab in the rabbit after intravitreal injection. We compared these values to those obtained using enzyme-linked immunosorbent assays (ELISA). Data obtained in this study were also compared to historical ranibizumab ocular PK data, either measured in-house or previously published. Three individual in vivo studies were performed in New Zealand White rabbits to assess the feasibility of using fluorophotometry to measure rabbit ocular PK of ranibizumab; explore the dynamic range of dosing fluorescently-labeled ranibizumab; and directly compare ranibizumab concentrations and calculated PK parameters measured by vitreal fluorophotometry to those measured using ELISA. In direct comparisons between fluorophotometry and ELISA, the calculated clearance (CL) values were 0.26 and 0.21 mL/day, the volumes of distribution at steady state (Vss) were 0.80 and 0.94 mL, the half-lives (t₁/₂) were 3.1 and 2.9 days and the dose normalized areas under the curve (AUC/D) were 4.7 and 3.9 μg·day/mL/μg, respectively. These values fell within the ranges of 0.13 to 0.44 mL/day for CL, 0.5 to 1.8 mL for Vss, 2.8 to 3.5 days for t1/2, and 2.3 to 7.9 μg·day/mL/μg for AUC/D that have been either measured previously in-house or published elsewhere. Although not suitable for measuring retinal concentrations, fluorophotometry is a valuable, noninvasive method to measure vitreous concentrations of protein therapeutics after intravitreal injection.
Fan Vincent C
Full Text Available Abstract Background Dry eye disease is a common debilitating ocular disease. Current diagnostic tests used in dry eye disease are often neither sensitive nor reproducible, making it difficult to accurately diagnose and determine end points for clinical trials, or evaluate the usefulness of different medications in the treatment of dry eye disease. The recently developed fluorophotometer can objectively detect changes in the corneal epithelium by quantitatively measuring its barrier function or permeability. The purpose of the study is to investigate the use of corneal fluorescein penetration measured by the fluorophotometer as a diagnostic tool in the evaluation of dry eye patients. Methods Dry eye patients (16 eyes, who presented with a chief complaint of ocular irritation corresponding with dry eye, low Schirmer's one test ( Results Ten minutes after fluorescein installition, patients with dry eye disease averaged a five-fold increase in corneal tissue fluorescein concentration (mean = 375.26 ± 202.67 ng/ml compared with that of normal subjects (mean = 128.19 ± 85.84 ng/ml. Sixty minutes after dye installation, patients with dry eye disease still revealed higher corneal tissue fluorescein concentration (mean = 112.87 ± 52.83 ng/ml compared with that of controls (mean = 40.64 ± 7.96 ng/ml, averaging a three-fold increase. Conclusion Patients with dry eye disease demonstrated an increased corneal permeability and a slower rate of elimination to topically administered fluorescein when measured by the fluorophotometer. This suggests that fluorophotometry may serve as a valuable quantitative and objective tool for the diagnosis of dry eye disease, and in following patients' response to new treatment modalities. Fluorophotometry may serve as an objective non-invasive tool for end-point analysis in clinical trials of new treatments for dry eye disease.
Fujitani, Kenji; Gadaria, Neha; Lee, Kyu-In; Barry, Brendan; Asbell, Penny
Diagnostic tests for dry eye disease (DED), including ocular surface disease index (OSDI), tear breakup time (TBUT), corneal fluorescein staining, and lissamine staining, have great deal of variability. We investigated whether fluorophotometry correlated with previously established DED diagnostic tests and whether it could serve as a novel objective metric to evaluate DED. Dry eye patients who have had established signs or symptoms for at least 6 months were included in this observational study. Normal subjects with no symptoms of dry eyes served as controls. Each eye had a baseline fluorescein scan prior to any fluorescein dye. Fluorescein dye was then placed into both eyes, rinsed with saline solution, and scanned at 5, 10, 15, and 30 min. Patients were administered the following diagnostic tests to correlate with fluorophotometry: OSDI, TBUT, fluorescein, and lissamine. Standard protocols were used. P eyes from 25 patients (DED = 22 eyes, 11 patients; Normal = 28 eyes, 14 patients) were included. Baseline scans of the dry eye and control groups did not show any statistical difference (p = 0.84). Fluorescein concentration of DED and normal patients showed statistical significance at all time intervals (p eyes up to 30 min after fluorescein dye instillation. There may be an aspect of DED that is missed in the current regimen of DED tests and only captured with fluorophotometry. Adding fluorophotometry may be useful in screening, diagnosing, and monitoring patients with DED.
Park, Minsung; Seo, Sungmin; Lee, In Su; Jung, Jong Hwa
A new fluorogenic based aminonaphthalimide-functionalized Fe(3)O(4)@SiO(2) core/shell magnetic nanoparticles 1 has been prepared, and its abilities to sense and separate metal ions were evaluated by fluorophotometry. The nanoparticles 1 exhibited a high affinity and selectivity for Hg(2+) and CH(3)Hg(+) ions over competing metal ions.
de Jong, C; Stolwijk, T; Kuppens, E; de Keizer, R; van Best, J
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.
Garaszczuk, Izabela K; Montes Mico, Robert; Iskander, D Robert; Expósito, Alejandro Cerviño
The aim is to provide a summary of methods available for the assessment of tear turnover and tear clearance rates. The review defines tear clearance and tear turnover and describes their implication for ocular surface health. Additionally, it describes main types of techniques for measuring tear turnover, including fluorescein tear clearance tests, techniques utilizing electromagnetic spectrum and tracer molecule and novel experimental techniques utilizing optical coherence tomography and fluorescein profilometry. Areas covered: Internet databases (PubMed, Science Direct, Google Scholar) and most frequently cited references were used as a principal resource of information on tear turnover rate and tear clearance rate, presenting methodologies and equipment, as well as their definition and implications for the anterior eye surface health and function. Keywords used for data-search were as follows: tear turnover, tear clearance, fluorescein clearance, scintigraphy, fluorophotometry, tear flow, drainage, tear meniscus dynamics, Krehbiel flow and lacrimal functional unit. Expert commentary: After decades, the topic of tear turnover assessment has been reintroduced. Recently, new techniques have been developed to propose less invasive, less time consuming and simpler methodologies for the assessment of tear dynamics that have the potential to be utilized in clinical practice.
Alvarez, Lawrence J.; Zamudio, Aldo C.; Candia, Oscar A.
Sildenafil citrate increases ocular blood flow and accelerates the rate of anterior chamber refilling after paracentesis. The latter effect could have resulted from a reduction in outflow facility or from an increase in aqueous humor (AH) production. In this study, we used scanning ocular fluorophotometry to examine the effects of sildenafil on AH turnover, and thus, AH production in eyes of live normal rabbits. For this, the rate of aqueous humor flow (AHF) was quantified with a commercially available fluorophotometer that measured the rate of fluorescein clearance from the anterior segment, which predominantly occurs via the trabecular meshwork. After ≈ 2 hrs of control scans to determine the baseline rate of AHF, the rabbits were fed 33 mg of sildenafil and allowed ≈ 45 min for the drug to enter the systemic circulation. Thereafter, fluorescence scans were retaken for an additional 90–120 min. Sildenafil ingestion increased AHF by about 36%, from 2.31 μL/min to 3.14 μL/min (PViagra, Revatio), stimulates AHF in rabbits. Our results seem consistent with reports indicating that the drug dilates intraocular arteries and augments intraocular vascular flow. These physiological responses to the agent apparently led to increased fluid entry into the anterior chamber. As such, the drug might have utility in patients with ocular hypotony resulting from insufficient AH formation. PMID:23562660
Motomizu, Shoji; Li, Zhen-Hai
Trace (phosphorus determination by flow-injection analysis are reviewed. Most of the methods cited in this review are fundamentally based on the reaction of orthophosphate with molybdate to form heteropoly acids, such as molybdenum yellow and molybdenum blue, and some of the methods are based on the formation of such secondary reactions as ion associates and their aggregates with bulky cations, such as cationic dyes and quaternary ammonium ions. The heteropoly acids themselves can be measured by spectrophotometry, and the ion associate formed with a cationic dye, Malachite Green (MG), can be measured based on the coloration of MG. Light scattering detection methods can be used for measuring the aggregates of ion associates formed with bulky cations. Highly sensitive detection of phosphorus can be accomplished by fluorophotometry; Rhodamine B (RB) and its analogues react with molybdophosphate to form ion associates, which shows fluorescence quenching of RB: LOD is about 5 nM. The detection method based on the chemiluminescence of luminal oxidized with molybdophosphoric acids is probably the most sensitive of all the detection methods reported so far: LOD of the method is as low as 1nM. The LOD of the molybdenum blue method can be improved by using a liquid core waveguide: LOD is 0.5 nM.
Muntz, Alex; Subbaraman, Lakshman N; Sorbara, Luigina; Jones, Lyndon
Tear exchange beneath a contact lens facilitates ongoing fluid replenishment between the ocular surface and the lens. This exchange is considerably lower during the wear of soft lenses compared with rigid lenses. As a result, the accumulation of tear film debris and metabolic by-products between the cornea and a soft contact lens increases, potentially leading to complications. Lens design innovations have been proposed, but no substantial improvement in soft lens tear exchange has been reported. Researchers have determined post-lens tear exchange using several methods, notably fluorophotometry. However, due to technological limitations, little remains known about tear hydrodynamics around the lens and, to-date, true tear exchange with contact lenses has not been shown. Further knowledge regarding tear exchange could be vital in aiding better contact lens design, with the prospect of alleviating certain adverse ocular responses. This article reviews the literature to-date on the significance, implications and measurement of tear exchange with contact lenses. Copyright © 2014 Spanish General Council of Optometry. Published by Elsevier Espana. All rights reserved.
J. L. Van Delft
Full Text Available One of the inflammatory responses of the eye to local application of platelet-activating factor (PAF is oedema of the conjunctiva, caused by extravasation of plasma. Aim of the study was to investigate if fluorescein would leak from the blood into the tears together with plasma protein after application of PAF to the eye. Fluorescein was given intraperitoneally 30 min prior to application of 25 μl of 0.1% solution of PAF. Thirty min after PAF the tear film was collected by washing the surface of the eye with 25 μl of phosphate buffered saline (PBS. Fluorescein in eye washings and in plasma was measured by fluorophotometry and albumin by immunodiffusion. Both fluorescein and albumin appeared in a related fashion in tears, being absent in washings of placebo-treated control eyes. Extravasation of fluorescein can be used as a measure for plasma leakage in the conjunctiva with the advantage over the Evans Blue method that the former is a non-invasive method.
Tian, Fengling; Huang, Wei; Yang, Jidong; Li, Qin
In pH 3.25-3.35 Britton-Robinson (BR) buffer solution, albendazole (ABZ) could react with eosin Y (EY) to form a 1:1 ion-association complex, which not only results in the quenching of fluorescence, but also resulted in the great enhancement of resonance Rayleigh scattering (RRS) and frequency doubling scattering (FDS). Furthermore, a new RRS spectrum will appear, and the maximum RRS wavelength was located at about 356 nm. The detection limit for ABZ were 21.51 ng mL-1 for the fluorophotometry, 6.93 ng mL-1 for the RRS method and 12.89 ng mL-1 for the FDS method. Among them, the RRS method had the highest sensitivity. The experimental conditions were optimized and effects of coexisting substances were evaluated. Meanwhile, the influences of coexisting substances were tested. The methods have been successfully applied to the determination of ABZ in capsules and human urine samples. The composition and structure of the ion-association complex and the reaction mechanism were discussed.
Alattar, M H; Ravindranath, T M; Choudhry, M A; Muraskas, J K; Namak, S Y; Dallal, O; Sayeed, M M
Sepsis-induced suppression in T-cell proliferation follows deranged Ca(2+) signaling in adult rats. In preliminary studies, we observed suppression in T-cell proliferation in septic neonatal rats as well. In this study, we assessed splenic T-cell cytosolic Ca(2+) concentration, [Ca(2+)](i), as its elevation plays an important role in T-cell proliferation. Also, we investigated the role of PGE(2) in sepsis-related changes in T-cell [Ca(2+)](i) in animals pretreated with cyclooxygenase-1 (COX-1) inhibitor (resveratrol) and cyclooxygenase-2 (COX-2) inhibitor (NS-398). Sepsis was induced in 15-day-old rat pups by intraperitoneal implantation of fecal pellets containing Escherichia coli and Bacteroides fragilis. The sham group consisted of pups implanted with sterile fecal pellets. Septic and sham pups were sacrificed 24 h after implantation and their spleens were removed. The spleens from sham and septic pups, along with spleens from unoperated control pups, were processed for single cell suspensions, and T cells were isolated using nylon wool columns. Fura-2 fluorophotometry was employed for the measurement of [Ca(2+)](i) (in nM units) in T cells stimulated with concanavalin A (ConA). Our results show that ConA-mediated T-cell [Ca(2+)](i) response is significantly suppressed in septic neonatal rats. Pretreatment of pups with COX-2, but not COX-1 inhibitor, prevented the decrease in the [Ca(2+)](i) response. These findings suggest that PGE(2) might induce the attenuation in T-cell Ca(2+) signaling during sepsis in neonatal rats. Copyright 2001 S. Karger AG, Basel
K.Yandrapu, Sarath; Upadhyay, Arun K.; Petrash, J. Mark; Kompella, Uday B.
Nanoparticles in porous microparticles (NPinPMP), a novel delivery system for sustained delivery of protein drugs, was developed using supercritical infusion and pressure quench technology, which does not expose proteins to organic solvents or sonication. The delivery system design is based on the ability of supercritical carbon dioxide (SC CO2) to expand poly(lactic-co-glycolic) acid (PLGA) matrix but not polylactic acid (PLA) matrix. The technology was applied to bevacizumab, a protein drug administered once a month intravitreally to treat wet age related macular degeneration. Bevacizumab coated PLA nanoparticles were encapsulated into porosifying PLGA microparticles by exposing the mixture to SC CO2. After SC CO2 exposure, the size of PLGA microparticles increased by 6.9 fold. Confocal and scanning electron microscopy studies demonstrated the expansion and porosification of PLGA microparticles and infusion of PLA nanoparticles inside PLGA microparticles. In vitro release of bevacizumab from NPinPMP was sustained for 4 months. Size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy, SDS-PAGE, and ELISA studies indicated that the released bevacizumab maintained its monomeric form, conformation, and activity. Further, in vivo delivery of bevacizumab from NPinPMP was evaluated using noninvasive fluorophotometry after intravitreal administration of Alexa Flour 488 conjugated bevacizumab in either solution or NPinPMP in a rat model. Unlike the vitreal signal from Alexa-bevacizumab solution, which reached baseline at 2 weeks, release of Alexa-bevacizumab from NPinPMP could be detected for 2 months. Thus, NPinPMP is a novel sustained release system for protein drugs to reduce frequency of protein injections in the therapy of back of the eye diseases. PMID:24131101
Jones, Michael P; Ward, Daniel A
To determine aqueous humor flow rate (AHFR) in an avian species by use of anterior segment fluorophotometry. 9 healthy red-tailed hawks (Buteo jamaicensis; 4 males and 5 females) that ranged from 8 months to 8 years of age. A protocol was developed for fluorophotometric determination of AHFR. Topical administration of 10% fluorescein was used to load the corneas, and corneal and aqueous humor fluorescein concentrations were measured approximately 5, 6.5, and 8 hours later. Concentration-versus-time plots were generated, and slopes and cornea-to-aqueous humor concentration ratios from these plots were used to manually calculate flow rates. Mean ± SD AHFRs for the right eye, left eye, and both eyes were 3.17 ± 1.36 μL/min (range, 1.67 to 6.21 μL/min), 2.86 ± 0.88 μL/min (range, 2.04 to 4.30 μL/min), and 2.90 ± 0.90 μL/min (range, 1.67 to 4.42 μL/min), respectively. The AHFRs were similar for right and left eyes. These flow rates represented a mean aqueous humor transfer coefficient of 0.0082/min, which is similar to that of mammalian species. The AHFR in red-tailed hawks was similar to that of most mammalian species, and the fractional egress was almost identical to that of other species. This information will allow a greater understanding of aqueous humor flow in avian eyes, which is crucial when evaluating diseases that affect avian eyes as well as medications that alter aqueous humor flow.
Yandrapu, Sarath K; Upadhyay, Arun K; Petrash, J Mark; Kompella, Uday B
Nanoparticles in porous microparticles (NPinPMP), a novel delivery system for sustained delivery of protein drugs, was developed using supercritical infusion and pressure quench technology, which does not expose proteins to organic solvents or sonication. The delivery system design is based on the ability of supercritical carbon dioxide (SC CO2) to expand poly(lactic-co-glycolic) acid (PLGA) matrix but not polylactic acid (PLA) matrix. The technology was applied to bevacizumab, a protein drug administered once a month intravitreally to treat wet age related macular degeneration. Bevacizumab coated PLA nanoparticles were encapsulated into porosifying PLGA microparticles by exposing the mixture to SC CO2. After SC CO2 exposure, the size of PLGA microparticles increased by 6.9-fold. Confocal and scanning electron microscopy studies demonstrated the expansion and porosification of PLGA microparticles and infusion of PLA nanoparticles inside PLGA microparticles. In vitro release of bevacizumab from NPinPMP was sustained for 4 months. Size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy, SDS-PAGE, and ELISA studies indicated that the released bevacizumab maintained its monomeric form, conformation, and activity. Further, in vivo delivery of bevacizumab from NPinPMP was evaluated using noninvasive fluorophotometry after intravitreal administration of Alexa Fluor 488 conjugated bevacizumab in either solution or NPinPMP in a rat model. Unlike the vitreal signal from Alexa-bevacizumab solution, which reached baseline at 2 weeks, release of Alexa-bevacizumab from NPinPMP could be detected for 2 months. Thus, NPinPMP is a novel sustained release system for protein drugs to reduce frequency of protein injections in the therapy of back of the eye diseases.
Full Text Available Ester Carreño1, Alejandro Portero2, David J Galarreta1,3, José M Herreras1,31Ocular Immunology Unit-IOBA (Instituto Universitario de Oftalmobiología, University of Valladolid, Campus Miguel Delibes, Valladolid, Spain; 2Ocular Immunology Unit, Hospital La Zarzuela, Madrid, Spain; 3Ocular Immunology Unit, Hospital Clínico Universitario de Valladolid, Valladolid, SpainAbstract: Ophthalmic bromfenac sodium sesquihydrate is a topically applied selective cyclooxygenase (COX-2 inhibitor. It is similar to amfenac, except for a bromine atom at the C4 of the benzoyl ring position, which markedly affects its in vitro and in vivo potency, extends the duration of anti-inflammatory activity, and enhances its inhibitory effect on COX-2 absorption across the cornea and penetration into ocular tissues. The United States Food and Drug Administration approved bromfenac in 2005 for the treatment of postoperative inflammation and the reduction of ocular pain in patients who have undergone cataract surgery. Nonsteroidal anti-inflammatory drugs (NSAIDs, and among them bromfenac, could be even more effective than steroids at reestablishing the blood–aqueous barrier, as revealed by flare on slit-lamp examination and as quantitatively measured using ocular fluorophotometry. Similar to other NSAIDs, it has a role in inhibiting intraoperative miosis during cataract surgery. However, bromfenac also seems to be useful in other situations, such as refractive surgery, allergic conjunctivitis (not useful in dry eye, choroidal neovascularization, and even ocular oncology. No reports of systemic toxicity have been published and bromfenac has good topical tolerance with a low incidence of adverse effects.Keywords: bromfenac, ophthalmic nonsteroidal anti-inflammatory drugs, inflammation, cataract surgery
. Analysis of diet samples was carried out using ICP-MS for Cs, Th and U, ICP-AES for Ca, Cu, Mg, Mn, Sr, INAA for Fe and Zn, ENAA for I, AAS for K, Na and Cd, fluoro-photometry for Se, and coldvapor- AAS for Hg
Lavery, W J; Kiel, J W
In humans, changing from upright to supine elicits an approximately 10 mmHg increase in cephalic venous pressure caused by the hydrostatic column effect, but episcleral venous pressure (EVP) and intraocular pressure (IOP) rise by only a few mmHg. The dissociation of the small increases in IOP and EVP compared to the larger increase in cephalic venous pressure suggests a regulatory mechanism controlling EVP. The aim of the present study was to determine if the rabbit model is suitable to study the effects of postural changes on EVP despite its short hydrostatic column. In anesthetized rabbits (n = 43), we measured arterial pressure (AP), IOP, and orbital venous pressure (OVP) by direct cannulation; carotid blood flow (BFcar) by transit time ultrasound, heart rate (HR) by digital cardiotachometer, and EVP with a servonull micropressure system. The goal of the protocol was to obtain measurement of supine EVP for ≈10 min, followed by ≈10 min of EVP measurement with the rabbit in a head down tilt. The data were analyzed by paired t-tests and the results reported as the mean ± standard error of the mean. In a separate group of animals (n = 35), aqueous flow was measured by fluorophotometry. This protocol entailed measurement of aqueous flow in the supine position for ≈60 min, followed by ≈60 min of aqueous flow measurement with the rabbit in a head down tilt. From supine to head down tilt, AP and BFcar were unchanged, IOP increased by 2.3 ± 0.4 mmHg (p measurements of the pressures and systemic parameters likely involved in the EVP responses to posture change. The present results indicate directionally similar EVP and IOP responses to tilt as occur in humans and, as in humans, the responses are smaller than would be expected from the change in the hydrostatic column height. Also, as in humans, the model reveals no change in aqueous flow during head down tilt. We conclude the rabbit model is appropriate for studying the mechanisms responsible for the relative