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Domain antibodies (dAb's) comprise the smallest functional unit of human IgG and can be targeted to a range of different soluble cytokine and receptor targets in the eye. In particular their small size may offer advantage for ocular tissue penetration and distribution. To investigate this we used a 13kDa tool molecule to undertake a preliminary short term ocular tissue distribution and pharmacokinetic study in the rabbit eye. The dAb was administered by the intravitreal or subconjunctival route or, as topical eye drops for up to five days and dAb concentrations measured in vitreous, aqueous, conjunctiva, choroid-RPE, retina, iris, sclera, and ciliary body. The observed elimination half-live of the dAb (~3 days) in vitreous showed a similar elimination rate to that of a much larger (∼50kDa) Fab fragment whilst the half-life following subconjunctival administration was ∼24 h and, after eye drop dosing the dAb was detectable in aqueous and conjunctiva. These preliminary data show that the intravitreal half-life of dAb's are similar to much larger antibody fragments, offering the potential to deliver significantly more drug to target on a molar basis with a single intravitreal injection potentially enabling dosing frequencies of once a month or less. Subconjunctival injection may provide short duration therapeutic levels of dAb to the anterior and posterior chamber whilst topical eye drop delivery of dAbs may be useful in front-of-eye disease. These data indicate that small domain antibodies may have utility in ophthalmology. Further studies are warranted., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition., Methods: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging., Results: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks., Conclusions: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Purpose: Development of myopia is associated with remodeling of the sclera, a tissue composed principally of collagen. Cyclic guanosine monophosphate (cGMP) regulates collagen synthesis in several organs; therefore, we investigated the effects of soluble guanylyl cyclase (sGC) stimulation and inhibition on refraction and ocular growth in guinea pigs under normal and form-deprived (FD) conditions., Methods: Retinal and scleral cGMP concentrations were measured in normal and monocularly FD guinea pigs at 2 days, 1 week, and 2 weeks of form deprivation and following 2 days recovery. Stimulation of sGC by BAY41-2272 and inhibition by NS-2028 were achieved by daily subconjunctival injection in normal and FD eyes. Refraction and axial parameters were measured at the commencement, middle, and cessation of the experiment. cGMP levels were also determined at the end of the experiment., Results: Retinal and scleral cGMP concentrations increased in FD eyes from 2 days to 2 weeks (P ≤ 0.029). Levels decreased after 2 days of recovery (P ≤ 0.003). Daily injections of BAY41-2272 induced a myopic shift (P ≤ 0.001) and ocular elongation (P ≤ 0.01) in normal animals, but did not alter myopia in FD eyes (P > 0.05). In contrast, daily injections of NS-2028 partially reduced myopic shifts (P ≤ 0.012) and ocular elongation (P ≤ 0.015) induced by form deprivation, but did not affect ocular growth and refraction in normal eyes (P > 0.05). Retinal and scleral cGMP levels were increased by BAY41-2272 in normal eyes and decreased by NS-2028 in FD eyes., Conclusions: Changes in cGMP signaling contribute to myopic development. Thus, cGMP may be a potential therapeutic target for preventing/treating myopia.

To study growth and development of ocular tissues, gene expression patterns in normal human fetal versus adult eyes were compared. Human retina/retinal pigment epithelium, choroid, sclera, optic nerve* and cornea* tissues were dissected from fetal (24 week gestational age) (N = 9; *N = 6), and adult (N = 6) normal donor eyes. The Illumina(®) whole genome expression microarray platform was used to assess differential expression. Statistical significance for all comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Significant gene expression fold changes > 1.5 were found in adult versus fetal retina/RPE (N = 1185), choroid (N = 6446), sclera (N = 1349), and cornea (N = 3872), but not optic nerve. Genes showing differential expression were assessed using Ingenuity Pathway Analysis (IPA) for enriched functions and canonical pathways. In all tissues, development, cell death/growth, cancer functions, and signaling canonical pathways were enriched. There was also a general trend of down-regulation of collagen genes in adult tissues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To determine the capacity and kinetics of the binding between triamcinolone acetonide (TA) and the ocular pigment for a better understanding of the transscleral delivery., Methods: In the in vitro study, natural melanin (sepia officinalis, Sigma-Aldrich) was incubated at 37°C with different concentrations of TA and the binding capacity/binding affinity was measured. The TA releasing profile from the melanin was also studied through repeated incubation of TA-melanin in fresh phosphate-buffed saline. In the ex vivo study, the effect of the choroidal pigment on the trans sclera/choroid permeability of TA was investigated through Franz-type vertical diffusion cells using both a TA suspension and a saturated TA solution., Results: The amount of TA bound to melanin increases with the increase of the TA concentration and with an increase in the incubation time. A Scatchard analysis revealed that the maximum number of moles of TA bound to melanin is predicted to be 22.43 nmol/mg, with a binding affinity of K=2.4×10(-5) nM(-1). TA released from a pigment showed a fast phase within the first 24 h and a slow phase thereafter. About 40% of the bound TA released in the first day and 73.94% of accumulative release was observed after 5 days. The TA suspension showed more TA penetration through the scleral-choroid complex than the saturated solution (P=0.0104). The apparent permeability coefficients for the suspension across the sclera-choroid of pigmented and albino rabbits are 7.48±1.53×10(-6) cm/s and 10.78±2.49×10(-6) cm/s, respectively., Conclusions: TA can bind to and release from the ocular pigment, which may extend the TA ocular half-life and therapeutic duration when TA is delivered through a subtenon injection. A further in vivo study is warranted to validate the findings and to quantitate the magnitude of the difference between pigmented and albino animals.

Since there is paucity of information on solute transporters in human ocular tissues, the aim of this study was immunohistochemical and functional characterization of peptide transporters (PEPT), organic cation transporters (OCTs), neutral and basic amino acid transporters (ATB(0,+)), and monocarboxylate transporters (MCTs) in human ocular barriers. Immunohistochemical localization of transporters was achieved using 5-µm-thick paraffin-embedded sections of whole human eyes. In vitro transport studies were carried out across human cornea and sclera-choroid-retinal pigment epithelium (SCRPE) using a cassette of specific substrates in the presence and absence of inhibitors to determine the role of transporters in transtissue solute delivery. Immunohistochemistry showed the expression of PEPT-1, PEPT-2, ATB(0,+), OCT-1, OCT-2, MCT-1, and MCT-3 in human ocular tissues. PEPT-1, PEPT-2, OCT-1, MCT-1, and ATB(0,+) expression was evident in the cornea, conjunctiva, ciliary epithelium, and neural retina. Expression of PEPT-1, PEPT-2, and OCT-1 was evident in choroid tissue as well. OCT-2 expression could be seen in the corneal and conjunctival epithelia, whereas MCT-3 expression was confined to the RPE layer. OCT-2 expression was evident in conjunctival blood vessel walls, whereas PEPT-1, PEPT-2, and OCT-1 were expressed in the choroid. Preliminary transport studies indicated inward transport of Gly-Sar (PEPT substrate), 1-methyl-4-phenylpyridinium (MPP+) (OCT substrate), and l-tryptophan (ATB(0,+) substrate) across cornea as well as SCRPE. For phenylacetic acid (MCT substrate), transporter-mediated inward transport across the cornea and outward transport across SCRPE were evident. Thus, PEPT, OCT, and ATB(0,+) are influx transporters present in human ocular barriers, and they can potentially be used for transporter-guided retinal drug delivery after topical, transscleral, and systemic administrations.

Purpose: Photodynamic therapy (PDT) laser light in conjunction with the benzoporphyrin derivative verteporfin is a current clinical treatment for choroidal vascular diseases such as age-related macular degeneration. The aim of this study was to examine the effects of PDT laser-activated and inactive verteporfin on various cultured ocular cells., Methods: Primary human scleral fibroblasts (hFibro), primary human trabecular meshwork (TM) cells (hTMC), primary porcine TM cells (pTMC), and a human retinal pigment epithelial cell line (ARPE-19 cells) were treated with verteporfin with and without activation by PDT laser. Cell viability was determined according to mitochondrial enzyme activity (3-(4,5- dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay)., Results: PDT laser treatment alone was insufficient to cause significant cell death in any of the cell types tested. Twenty-four-hour exposure to inactive verteporfin (without PDT laser) caused a dose-dependent decrease in cell viability in hFibro and hTMC, and to a lesser extent ARPE-19 cells. Verteporfin (0.5 µg/ml) without PDT laser activation caused a slight but statistically insignificant reduction in cell viability in hFibro (81.5% ± 19.3%), pTMC (82.9% ± 6.7%), hTMC (80.3% ± 7.7%), and ARPE-19 cells (84.5% ± 14.9%). Verteporfin (0.5 µg/ml) plus 50 µJ/cm(2) PDT laser treatment significantly decreased viability in hFibro (13.5% ± 3.3%), pTMC (7.1% ± 1.5%), hTMC (11.1% ± 5.2%), and ARPE-19 (44.5% ± 7.8%). Similar results were obtained in cells where verteporfin incubation was followed by washout before PDT laser, indicating that verteporfin is internalized by the studied cell lines., Conclusions: PDT laser-induced cell death was obtained with coincubation of verteporfin or preincubation followed by washout. These results suggest a potential future use of PDT therapy for selective in vivo removal of targeted ocular cells beyond the current use for destroying vascular endothelial cells.

To treat ophthalmic diseases like glaucoma or inflammatory disorders topically applied ophthalmic formulations such as eye drops are usually used. In addition, novel ophthalmic implants releasing drug substances locally into different parts of the eye are available today. In the work presented here, the permeability coefficients of selected drugs (ciprofloxacin hydrochloride, lidocaine hydrochloride, timolol maleate) for ophthalmic tissues were determined using side-by-side diffusion chambers (so-called Ussing chambers). Sclera, conjunctiva, cornea, choroidea-retina-complex and a complex of conjunctiva-sclera-choroidea-retina were excised from fresh porcine, rabbit and bovine eyes. In the porcine eye tissues the highest P(app) values were obtained for conjunctiva with the exception of lidocaine. Therefore, it can be estimated that a certain amount of drug diffuses or is transported through conjunctiva after application. The P(app) values for sclera were also higher than those for cornea and even more, the surface area of sclera which is available for drug absorption is much larger than that of cornea when applying an implant. The obtained permeability coefficients for sclera and conjunctiva indicate that the administration of periocular implants can be an alternative to topically applied formulations. The complexes of the tissues were a significantly (p<0.01) stronger barrier to the investigated substances than the separated tissues. Distinct differences in permeability coefficients between the investigated animal tissues were observed. Overall the highest P(app) values for all mounted tissues were obtained with the rabbit, followed by porcine and bovine eyes. Because of these distinct interspecies differences one must be very careful when selecting the proper animal model for the permeability experiments., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The pharmacologically active compound (33% reduction in rabbit intraocular pressure recovery rate assay) 1-ethyl-6-fluoro-1,2,3,4-tetrahydroquinoline (MC4), which showed ocular hypotensive action and had optimum physicochemical properties, was characterized for its ocular absorption and distribution properties to better understand its in vivo potency in comparison with an inactive compound, N-ethyl-1,4-benzoxazine (MC1). Tissue distribution to various ocular tissues was determined after absorption by both corneal and conjunctival-scleral routes, following administration by the "topical infusion" technique. The rank order of penetration for both the compounds was cornea > iris-ciliary body > aqueous humor > lens > conjunctiva-sclera. Overall, MC4 had significantly higher concentrations than MC1 in various ocular tissues, but particularly in the iris-ciliary body, which is the site of action (biophase). Ocular disposition studies of the active compound MC4 were then conducted to characterize its elimination kinetics, and the pharmacokinetic parameters were determined by non-compartmental and moment analysis using equations specific to "topical infusion" technique: first-order absorption rate constant, 4.1 × 10(-4) min(-1) ; elimination rate constant, 0.012 min(-1) ; mean residence time, 39.6 min; steady-state volume of distribution, 0.721 mL; and aqueous humor ocular clearance, 8.44 µL/min. The results were consistent with the conclusion that MC4 is well absorbed and distributed to the active site., (Copyright © 2011 Wiley-Liss, Inc.)

Purpose: The authors applied a novel microindentation technique to characterize biomechanical properties of small ocular and orbital tissue specimens using the hertzian viscoelastic formulation, which defines material viscoelasticity in terms of the contact pressure required to maintain deformation by a harder body., Methods: They used a hard spherical indenter having 100 nm displacement and 100 μg force precision to impose small deformations on fresh bovine sclera, iris, crystalline lens, kidney fat, orbital pulley tissue, and orbital fatty tissue; normal human orbital fat, eyelid fat, and dermal fat; and orbital fat associated with thyroid eye disease. For each tissue, stress relaxation testing was performed using a range of ramp displacements. Results for single displacements were used to build quantitative hertzian models that were, in turn, compared with behavior for other displacements. Findings in orbital tissues were correlated with quantitative histology., Results: Viscoelastic properties of small specimens of orbital and ocular tissues were reliably characterized over a wide range of rates and displacements by microindentation using the hertzian formulation. Bovine and human orbital fatty tissues exhibited highly similar elastic and viscous behaviors, but all other orbital tissues exhibited a wide range of biomechanical properties. Stiffness of fatty tissues tissue depended strongly on the connective tissue content., Conclusions: Relaxation testing by microindentation is a powerful method for characterization of time-dependent behaviors of a wide range of ocular and orbital tissues using small specimens, and provides data suitable to define finite element models of a wide range of tissue interactions.

The influence of drug properties including solubility, lipophilicity, tissue partition coefficients, and in vitro transscleral permeability on ex vivo and in vivo transscleral delivery from corticosteroid suspensions was determined. Solubility, tissue/buffer partition coefficients for bovine sclera and choroid-retinal pigment epithelium (CRPE), and in vitro bovine sclera and sclera-choroid-retinal pigment epithelium (SCRPE) transscleral transport were determined at pH 7.4 for triamcinolone, prednisolone, dexamethasone, fluocinolone acetonide, triamcinolone acetonide, and budesonide in solution. Ex vivo and in vivo transscleral delivery was assessed in Brown Norway rats after posterior subconjunctival injection of a 1 mg/ml suspension of each corticosteroid. Corticosteroid solubility and partition coefficients ranged from ∼ 17 to 300 μg/ml and 3.0 to 11.4 for sclera and from 7.1 to 35.8 for CRPE, respectively, with the more lipophilic molecules partitioning more into both tissues. Transport across sclera and SCRPE was in the range of 3.9 to 10.7% and 0.3 to 1.8%, respectively, with the transport declining with an increase in lipophilicity. Ex vivo and in vivo transscleral delivery indicated tissue distribution in the order CRPE ≥ sclera > retina > vitreous. Tissue partitioning showed a positive correlation with drug lipophilicity (R(2) = 0.66-0.96). Ex vivo and in vivo sclera, CRPE, retina, and vitreous tissue levels of all corticosteroids showed strong positive correlation with drug solubility (R(2) = 0.91-1.0) but not lipophilicity (R(2) = 0.24-0.41) or tissue partitioning (R(2) = 0.24-0.46) when delivered as suspensions. In vivo delivery was lower in all eye tissues assessed than ex vivo delivery, with the in vivo/ex vivo ratios being the lowest in the vitreous (0.085-0.212). Upon exposure to corticosteroid suspensions ex vivo or in vivo, transscleral intraocular tissue distribution was primarily driven by the drug solubility.

The purpose of this study was to analyze drug pharmacokinetics in the posterior eye tissues after topical instillation. For the in vivo study, the concentrations of ofloxacin in rabbit ocular tissues were analyzed by high performance liquid chromatography at 1, 2, and 3 h after instillation. For the in silico simulation, the concentration distribution of ofloxacin in the eye was calculated by the ocular pharmacokinetic model based on the diffusion/partition model. The simulated profiles were then compared with the in vivo experimental findings. In the in vivo study, the drug concentration in the posterior vitreous body initially decreased with time after topical instillation, and thereafter, the concentration increased. The in silico simulation of ocular pharmacokinetics indicated that the drug penetration of the posterior vitreous body was determined by three major pathways: (1) the initial transscleral penetration, (2) the intermediate transcorneal penetration, and (3) the late transretinal penetration. The in vivo findings were well described by a series of contributions by these three pathways. In conclusion, the present in vivo and in silico studies suggest that the instilled drugs initially reached the posterior vitreous body by diffusion through the sclera and then later by corneal penetration and systemic circulation.

Purpose: The selective muscarinic antagonist pirenzepine inhibits experimentally induced myopia in avian and mammalian species, including nonhuman primates and adolescent humans. Transient positive lens defocus has a potent inhibitory effect on negative-lens-induced myopia in avian and mammalian models. The purpose of the present study was to determine the influence of daily treatment with pirenzepine on ocular growth and refractive error in chicks wearing positive lenses., Methods: The chicks were allocated to one of eight groups (n = 6 each group) on the basis of whether they wore +10 or -10 D lenses monocularly and whether they received daily intravitreal injections of pirenzepine (700 μg) or vehicle (phosphate-buffered saline) in the lens-defocused eye. In vivo refractive and biometric data were collected, and glycosaminoglycan synthesis in the sclera was assessed., Results: Pirenzepine did not alter the level of positive-lens-induced hyperopia in chicks wearing +10 D lenses compared with that in the vehicle control group (+8.1 ± 0.6 D vs. +8.9 ± 2.4 D, mean ± SEM; P = 0.76). In contrast, pirenzepine caused significant inhibition of negative-lens-induced myopia compared with that in the vehicle group (-1.1 ± 1.5 D vs. -8.8 ± 1.1 D; P = 0.001). Glycosaminoglycan synthesis in the posterior sclera was significantly increased in the negative-lens-treated groups and showed small decreases in the positive-lens-treated groups., Conclusions: The influence of pirenzepine on ocular growth in chicks differed by sign of lens defocus, with pirenzepine blocking negative-lens effects on ocular growth, but not positive-lens effects. The most likely reason that hyperopia was not enhanced by pirenzepine treatment was that the rapid compensatory eye growth associated with positive lenses eliminated the imposed myopic defocus, and the clear retinal image prevented any additional hyperopia from developing.

A diffusion model of ocular pharmacokinetics was used to estimate the effects of pathological conditions on ocular pharmacokinetics. In vivo rabbit data after topical instillation of ciprofloxacin and ofloxacin were compared with the simulated concentrations in the aqueous and vitreous humors. The barrier capacity of the surrounding membranes such as the retina/choroid/sclera (RCS) membrane and the cornea was characterized by dimensionless Sherwood number derived by the pseudo-steady state approach (PSSA). We assumed the barrier capacity decreased by inflammation; when the barrier capacity of the RCS membrane and the cornea was assumed to be one-tenth for the RCS membrane and a half for the cornea respectively, the in vivo data agreed with the simulated profile without contradiction. The drug concentration gradient simulated in the vitreous body near the RCS membrane was more significant in the inflamed eyes than in the normal eyes, suggesting that the elimination of the drugs from the RCS membrane was enhanced by inflammation. The present diffusion model can better describe the ocular pharmacokinetics in both normal and diseased conditions.

Recently we showed a critical role for Vascular Adhesion Protein-1 (VAP-1) in rodents during acute ocular inflammation, angiogenesis, and diabetic retinal leukostasis. However, the expression of VAP-1 in the human eye is unknown. VAP-1 localization was therefore investigated by immunohistochemistry. Five micrometer thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5 microg/ml), smooth muscle actin (1 microg/ml), CD31 or isotype-matched IgG at 4 degrees C. Subsequently, a secondary mAb was used for 30 min at room temperature, followed by Dako Envision + HRP (AEC) System for signal detection. The stained sections were examined using light microscopy and the signal intensity was quantified by two evaluators and graded into 4 discrete categories. In all examined ocular tissues, VAP-1 staining was confined to the vasculature. VAP-1 labeling showed the highest intensity in both arteries and veins of neuronal tissues: retina and optic nerve, and the lowest intensity in the iris vasculature (p < 0.05). Scleral and choroidal vessels showed moderate staining for VAP-1. VAP-1 intensity was significantly higher in the arteries compared to veins (p < 0.05). Furthermore, VAP-1 staining in arteries colocalized with both CD31 and smooth muscle actin (sm-actin) staining, suggesting expression of VAP-1 in endothelial cells, smooth muscle cells or potentially pericytes. In conclusion, immunohistochemistry reveals constitutive expression of VAP-1 in human ocular tissues. VAP-1 expression is nearly exclusive to the vasculature with arteries showing significantly higher expression than veins. Furthermore, VAP-1 expression in the ocular vasculature is heterogeneous, with the vessels of the optic nerve and the retina showing highest expressions. These results characterize VAP-1 expression in human ocular tissues.

The distribution and clearance of macromolecules in ocular delivery are not well understood. It has been hypothesized that iontophoresis can enhance transscleral delivery of macromolecules. The objective of this study was to investigate the ocular distribution of a macromolecule after transscleral iontophoretic delivery and intravitreal injection in vivo using nuclear magnetic resonance imaging (MRI) and to compare these results. Experiments of constant current transscleral iontophoresis of 4mA or intravitreal injection were performed on New Zealand white rabbits in vivo. Iontophoresis experiments were also performed on rabbits postmortem. Galbumin (Gd-labeled albumin) was the model permeant surrogate to clinical therapeutic agents. MRI was used to monitor the distribution of the molecule in the eye after ocular iontophoresis and intravitreal injection. In addition, the conjunctiva, sclera, choroid, and retina were extracted in the transscleral iontophoresis study to determine the amounts of Galbumin in these tissues using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The results show that iontophoresis enhanced the ocular delivery of Galbumin. The macromolecule was mainly delivered into the conjunctiva and sclera in microgram quantities and then diffused towards the posterior section in the upper hemisphere of the eye in vivo. Both in vivo and postmortem studies show that the iontophoretic delivery of Galbumin into the vitreous was below the detection limit. In the intravitreal injection study, the diffusion coefficient of Galbumin in the vitreous humor was estimated to be close to that of free aqueous diffusion.

UV-induced oxidation damage seems to play a major role in a number of specific pathological conditions of intraocular tissues, such as cataract formation and retinal degeneration. Therefore, antioxidant and/or scavenger compounds might protect the eyes from UV-induced cellular damage. We previously reported that 4-coumaric acid (4-CA) is able to protect rabbit corneal-derived cells (SIRC) from UVB-induced oxidation damage. In this study we evaluated the protective effect of 4-CA against UVB-induced cell damage in rabbit cornea in vivo. Twelve male New Zealand albino rabbits were used; four rabbits were used as a control and received vehicle in one eye and 4-CA acid in the contralateral eye; eight rabbits were exposed to UVB rays (79.2mJ/cm(2)) and three days before to UV exposure each animal received 1 drop/day of vehicle in one eye and 1 drop/day of vehicle containing 4-CA (164ng) in the contralateral eye. Corneal and sclera tissues were removed and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels were measured. Superoxide dismutase (SOD) and xanthine oxidase (XO) activities were determined in aqueous humour. UVB-induced vessel hyper-reactivity was strongly reduced at 4 and 24h after UVB exposure after local treatment with 4-CA, 8-oxodGuo levels, a marker of oxidative DNA damage, were significantly increased (P<0.05) in sclera and cornea by UVB irradiation, but when 4-CA was administered to the conjunctiva in a buffered solution once a day for 3d before and 6d after UVB exposure, levels of 8-oxodGuo were similar to controls and significantly reduced (P<0.05) compared to UVB-treated corneas. XO activity in the aqueous humour was significantly increased. The administration of 4-CA for 3d before and 6d after UVB irradiation induced a small but significant (P<0.05) reduction of XO compared with control eyes. Our results indicate that the administration of 4-CA protects eye tissues, thus reducing the harmful effect of UVB radiation at low concentration, probably through its free radical scavenging and antioxidant properties. Therefore, 4-CA may be useful in protecting the eye from free radical damage following UVB exposure from sunlight, UV lamps and welding torches.

Purpose: Several studies suggest that postnatal ocular growth is under the control of factors within the eye that regulate the rate of scleral extracellular matrix remodeling and the rate of ocular elongation. A microarray analysis was employed to identify some of the factors involved in the regulation of visually guided ocular growth. Gene expression was compared in the retina-retinal pigmented epithelium (RPE)-choroid of chick eyes that were decelerating in the rate of ocular growth ("recovering" from myopia) as compared with contralateral control eyes., Methods: Form-deprivation myopia was induced in the right eyes of two-day-old chicks by the application of translucent occluders. Following 10 days of deprivation, occluders were removed and chicks were provided unrestricted vision for an additional 1-7 days (recovery). After one and four days of recovery, chicks were sacrificed, retina, RPE, and choroid were isolated, and mRNA was subjected to microarray analysis using a chicken immune system 4000 gene microarray. In addition, whole eyes and isolated ocular tissues (retina and RPE, choroid, sclera, and extraocular muscle) of treated and control eyes were subjected to real-time PCR, immunohistochemistry, and western blot analyses to verify gene expression results., Results: Following one day of recovery, only one gene, avian thymic hormone (ATH) was highly upregulated (+12.3 fold). ATH gene and protein expression were confirmed in the retina and choroid as well as in the sclera and extraocular muscle. A significant increase in ATH protein was detected in choroids from treated eyes following four days of recovery as compared to contralateral controls (p<0.05; Wilcoxon signed-rank test)., Conclusions: ATH is expressed in several ocular tissues and is specifically and rapidly (within one day) upregulated in the choroids of chick eyes recovering from induced myopia. This upregulation corresponds to the onset of choroidal thickening and increased choroidal vascular permeability. The identification of ATH in ocular tissues and its increased protein accumulation in the choroid during recovery from induced myopia suggest a novel role for this protein in the choroidal response to myopic defocus.

Purpose: Chick eyes compensate for the defocus imposed by positive or negative spectacle lenses. Glucagon may signal the sign of defocus. Do insulin (or IGF-1) and glucagon act oppositely in controlling eye growth, as they do in metabolic pathways and in control of retinal neurogenesis?, Methods: Chicks, wearing lenses or diffusers or neither over both eyes, were injected with glucagon, a glucagon antagonist, insulin, or IGF-1 in one eye (saline in the other eye). Alternatively, chicks without lenses received insulin plus glucagon in one eye, and either glucagon or insulin in the fellow eye. Ocular dimensions, refractive errors, and glycosaminoglycan synthesis were measured over 2 to 4 days., Results: Glucagon attenuated the myopic response to negative lenses or diffusers by slowing ocular elongation and thickening the choroid; in contrast, with positive lenses, it increased ocular elongation to normal levels and reduced choroidal thickening, as did a glucagon antagonist. Insulin prevented the hyperopic response to positive lenses by speeding ocular elongation and thinning the choroid. In eyes without lenses, both insulin and IGF-1 speeded, and glucagon slowed, ocular elongation, but glucagon and insulin each increased the rate of thickening of the crystalline lens. When injected together, insulin blocked choroidal thickening by glucagon, at a dose that did not, by itself, thin the choroid., Conclusions: Glucagon and insulin (or IGF-1) cause generally opposite modulations of eye growth, with glucagon mostly increasing choroidal thickness and insulin mostly increasing ocular elongation. These effects are mutually inhibitory and depend on the visual input.

This paper studied the effect on UV-B ocular damage of 10microM hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5 microM HCAF+5 microM p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m(2)) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m(2)). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E(2) (PGE(2)) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.

Aim: The aim of this study was to evaluate methylprednisolone penetration into ocular structures after low-current trans-scleral hydrogel iontophoresis, as compared with the common intravenous (i.v.) treatment., Methods: Methylprednisolone hemisuccinate (MPH) iontophoresis was studied in rabbits, using drug-loaded hydrogels mounted on a portable iontophoretic device. Cathodal iontophoresis of 2.6 mA/cm(2) was applied for 5 min at two opposite sites on the sclera or for 10 min at the same site. Ocular drug levels were determined 2 h after iontophoretic treatment, then compared to mock iontophoresis and i.v. infusion of 10 mg/kg methylprednisolone., Results: Significantly higher methylprednisolone levels were found in ocular tissues after iontophoresis, compared with the control groups, except for the sclera concentrations, which were similar to the concentrations achieved after mock iontophoresis. Two (2) h after the trans-scleral iontophoretic treatment, 178.59 +/- 21.63 microg/g, 6.74 +/- 2.38 microg/ml, and 2.71 +/- 0.57 microg/mL were found in the retina, aqueous humor, and vitreous, respectively. No significant differences were found between one or two site treatments of trans-scleral iontophoresis. Nondetectable concentrations were found 2 h after the i.v. infusion of 10 mg/kg of methylprednisolone in all evaluated ocular tissues and fluids., Conclusions: A short, low-current noninvasive iontophoretic treatment, using methylprednisolone-loaded hydrogels, has potential clinical value in treating ocular inflammatory diseases.

Purpose: To evaluate the efficiency of the dexamethasone phosphate penetration into the rabbit eye after transcorneal and transscleral iontophoresis using a drug loaded hydrogel assembled on a portable iontophoretic Mini Ion device., Methods: lontophoresis of dexamethasone phosphate was studied in healthy rabbits using drug-loaded disposable HEMA hydrogel sponges and portable iontophoretic device. Corneal iontophoretic administration was performed with electric current of 1 mAmp for 1, 2, and 4 min. In the control group, the dexamethasone was applied in drops into the conjunctival sac. Transconjunctival and transscleral iontophoresis were performed in the pars plana area, through the conjunctiva or directly on the sclera. Dexamethasone concentrations were assayed using HPLC method. To study the anatomical changes after iontophoresis application, histological examinations of corneas excised 5 minutes and 8 hours after the procedure were performed., Results: Dexamethasone levels in the rabbits' corneas after a single transcorneal iontophoresis were up to 38 times higher compared to those obtained after topical eye drops instillation. High drug concentrations were obtained in the retina and sclera 4 hours after transscleral iontophoresis as well. There were no statistically significant differences in the drug concentration after transscleral and tranconjunctival iontophoresis. Histological examination of the corneas after the iontophoresis showed only discrete reversible changes of the epithelium and the stroma., Conclusion: A short, low-current, non-invasive iontophoretic treatment using the dexamethasone-loaded hydrogels has a potential clinical value in increasing the drug's penetration into the anterior and posterior segment of the eye.

Cystinosis is a lysosomal storage disorder characterized by abnormal accumulation of cystine, which forms crystals at high concentrations. The causative gene CTNS encodes cystinosin, the lysosomal cystine transporter. The eye is one of the first organs affected (corneal lesions and photophobia in the first and visual impairment in the second decade of life). We characterized the ocular anomalies of Ctns-/- mice to determine whether they mimic those of patients. The most dramatic cystine accumulation was seen in the iris, ciliary body, and cornea of Ctns-/- mice. Consistently, Ctns-/- mice had a low intraocular pressure (IOP) and seemed mildly photophobic. Retinal cystine levels were elevated but increased less dramatically with age. Consistently, the retina was intact and electroretinogram (ERG) profiles were normal in mice younger than 19 mo; beyond this age, retinal crystals and lesions appeared. Finally, the lens contained the lowest cystine levels and crystals were not seen. The temporospatial pattern of cystine accumulation in Ctns-/- mice parallels that of patients and validates the mice as a model for the ocular anomalies of cystinosis. This work is a prerequisite step to the testing of novel ocular cystine-depleting therapies.

It is generally accepted that the increase in choroidal thickness in response to myopic defocus in chicks acts to move the retina towards the image plane. It may also constitute part of the signal cascade in the visual regulation of eye growth. To test this, we used the nitric oxide synthase inhibitor l-NAME to inhibit the defocus induced choroidal thickening under two different visual conditions, and looked at the effects on ocular growth rate. Exp. 1: Deprivation/Vision: chicks were monocularly deprived of form vision with translucent diffusers from day 6 to day 9. In the middle of each day the diffusers were removed for 2 h. One group received an intravitreal injection of 30 microl l-NAME (16 micromole; n=12) prior to the vision, a second group received injections of physiological saline (n=11). Exp. 2: Recovery/Vision: chicks were made myopic by form deprivation from day 6 to day 10. On days 11 to 14 the diffusers were removed for 2 h per day for 4 days to allow eyes to "recover" from the myopia. One group received an injection of l-NAME prior to vision (n=8), the other saline (n=6). Refractive errors were measured with a refractometer at the start (days 6 and 11) and end (days 10 and 15, respectively) of both experiments. Ocular dimensions were measured with high frequency A-scan ultrasonography at the start and end, and on the third experimental day immediately before and after the period of vision. Choroidal retinoic acid synthesis was measured by HPLC. Finally, NO production and scleral proteoglycan synthesis were measured in eyes wearing positive lenses 6 and 24h after an injection of l-NAME. l-NAME prevented the transient vision-induced choroidal thickening in both experiments. Furthermore, l-NAME inhibited the protective effect of brief daily vision: eyes became significantly more myopic than saline controls (exp. 1: -9 D vs -2.7D; exp. 2: -0.9 D vs +4.3 D; p<0.005 for both) and grew faster (change in lens-sclera: exp. 1: 295 vs 158 microm; exp. 2: 147 vs 39 microm; p<0.05 for both). Notably, l-NAME inhibited the growth of the anterior chamber (exp. 1: 88 vs 185 microm; exp. 2: 147 vs 254 microm; p<0.01 for both). Injections of l-NAME after the periods of vision had no effect on growth at the back of the eye, but still had an inhibitory effect on the anterior chamber. Retinoic acid levels in the drug-injected choroids were significantly decreased compared to controls. In eyes responding to positive lenses, l-NAME inhibited NO synthesis and disinhibited scleral glycosaminoglycan synthesis 6h after the injection. In summary, preventing the transient vision-induced increases in choroidal thickness altered ocular growth rate in a consistent manner under two different visual conditions, in both preventing the vision-induced reduction in growth rate. This supports the hypothesis that visually-induced changes in choroidal thickness play a role in the visual regulation of ocular growth.

Anthocyanins (ACs) are phenolic compounds that are distributed widely in fruits and vegetables. Although consumption of these compounds has been shown to improve visual function, the distribution of ACs in ocular tissue has not been examined in detail. The aim of this study was therefore to evaluate the ocular distribution of blackcurrant anthocyanins (BCAs) in rats and rabbits after oral, intravenous (i.v.) and intraperitoneal (i.p.) administration. Identification and quantification of ACs were carried out using high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and high-performance liquid chromatography (HPLC) with UV-visible detection, respectively. BCAs were identified in the plasma and whole eye after oral and i.p. administration in rats. No other peaks were detected in either plasma or ocular tissues after administration when the absorbance of the eluate was monitored at 520 nm. This finding indicates that intact forms of ACs were present in rats after administration of BCA. In rats given i.p. administration, the concentration of total ACs in the whole eye and some ocular tissues was higher than that measured in plasma. These results suggested that ACs detected in the ocular tissues were not due to residual blood. Following i.v. administration in rabbits, four ACs were identified in the plasma and several ocular tissues including the aqueous humor, cornea, sclera, choroid, ciliary body, iris and retina. A small amount of ACs was also detected in the vitreous and lens. In conclusion, this study demonstrated that BCAs were absorbed and distributed in ocular tissues as intact forms. Our data show clearly that intact forms of BCAs pass thorough the blood-aqueous barrier and blood-retinal barrier in both rats and rabbits.

Purpose: Ocular development involves changes in extracellular matrix components of the scleral wall as it expands. This study was conducted to determine scleral gene expression profiles during mouse ocular development to identify genes involved in normal scleral growth., Methods: Sample sets of pooled sclerae of 3- and 8-week-old mice were microdissected, and total RNA was isolated. After reverse transcription, the cDNA was in vitro transcribed to produce biotin-labeled cRNA. The purified biotin-labeled cRNA samples were hybridized to microarray chips (GeneChip Mouse Genome 430 2.0; Affymetrix, Santa Clara, CA). Gene transcript expression profiles were determined, and eight differentially expressed genes between the two age groups underwent further confirmation by real-time PCR analysis., Results: Differential regulation of 4884 gene transcripts in mouse sclera with less than 5% false-discovery rate (FDR) was identified. The top 1000 with the lowest FDR among the 4884 probe sets were filtered for threefold changes between the two age groups, and 718 gene transcripts were identified. Among these 718 gene transcripts, 210 were upregulated and 508 downregulated in adult relative to juvenile mouse sclera. TGF-beta1 and several collagen genes were significantly downregulated. Microarray differential expression by real-time PCR validation of eight extracellular matrix-associated gene transcripts was confirmed., Conclusions: This is the first study to demonstrate gene expression profiles in mouse sclera during ocular growth. These findings support the role of TGFbeta1 as a signaling molecule in modulating extracellular matrix during ocular development. This endeavor may be helpful in furthering understanding of how scleral remodeling is regulated during eye growth.

Purpose: The influences of diurnal rhythms involving a variety of ocular parameters are implicated in the development of myopia. The purpose of this study was to determine the expression of the melatonin receptor subtype proteins in chick ocular tissues and to examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia., Methods: Expression of the Mel(1a), Mel(1b), and Mel(1c) melatonin receptor proteins in ocular tissues was examined by Western blot analyses, slot blot analyses, and immunocytochemistry. For examining the effect of melatonin on ocular growth, chicks were maintained on a 12-hour light-dark cycle and were monocularly form-vision deprived in one eye with a translucent occluder for 5 days. During the 5-day treatment period, chicks were injected systemically during the early dark period with melatonin (0.6 mg) or 2% ethanol vehicle control. Ocular dimensions of normal and deprived eyes were examined by high frequency A-scan ultrasound., Results: Immunocytochemical analysis of chick ocular tissues revealed the cellular distribution of the Mel(1a) receptor subtype in the cornea, choroid, sclera, and retina. Western blot and slot blot analyses demonstrated that all three receptors were present in these tissues and they demonstrated distinct diurnal rhythms of protein expression in the retina-RPE-choroid, with peak levels of Mel(1a) and Mel(1b) occurring during the night and peak levels of Mel(1c) during the day. Systemic administration of melatonin resulted in significant changes in anterior chamber depth, vitreous chamber depth, and choroidal thickness of form-deprived and/or control eyes., Conclusions: Results of this study show that all three melatonin receptor subtypes are expressed in retinal and extraretinal ocular tissues of the chick eye. The finding that administration of melatonin alters the growth of several ocular tissues in both control and form-deprived eyes suggests that melatonin, acting through specific melatonin receptors in ocular tissues, plays a role in ocular growth and development. This conclusion suggests that the action of melatonin, combined with expression of melatonin receptors, is involved in the regulation of the diurnal rhythm of ocular growth.

We investigated whether poly-L-arginine (PLA) could improve permeation of the hydrophilic compounds, FITC-labeled dextran (MW 3800, FD-4) and pyridoxamine, through ocular surface tissues. Samples of cornea, conjunctiva, or conjunctiva/sclera composite from Japanese white rabbits were mounted in Ussing chambers to measure FD-4 and pyridoxamine permeation and transepithelial electric resistance (TEER). The integrity and viability of the conjunctiva were assessed by chronological TEER monitoring and MTT assay, respectively. The permeability coefficient (Papp) of FD-4 in the cornea, conjunctiva, and conjunctiva/sclera composite was increased by the addition of PLA (MW 38 kDa, PLA (50) at 0.1 mg/ml by 6.81-, 9.78-, and 7.91-fold, respectively. The Papp of pyridoxamine was also increased in the presence of PLA (50) by 7.98-, 4.67-, 8.31-fold, respectively. A corresponding reduction in TEER was observed in all tissues. However, the reduced TEER in the case of the conjunctiva had recovered to ca. 70% 120 min after replacing the mucosal fluid with fresh bicarbonated Ringer's solution. MTT assay results indicated that treatment of the conjunctiva with 0.1 mg/ml PLA (50) did not change the production of formazan compared to that without PLA (50), indicating that the conjunctival viability is not significantly affected by PLA (50). Our findings suggest that PLA may be useful in promoting the ocular delivery of hydrophilic drugs without producing significant epithelial damage.

Purpose: To investigate the effect and safety of benzalkonium chloride on transscleral drug delivery in the rabbit after continuous intrascleral administration., Methods: Betamethasone 21-phosphate (BP) aqueous solutions, with or without benzalkonium chloride (BAK), were continuously administered to albino rabbit sclera with an osmotic pump for 1 week. The BP concentrations in the aqueous humor, vitreous, and retina-choroid were measured by high-performance liquid chromatography (HPLC). To investigate the effect of BAK on scleral permeability of BP in vitro, penetration of BP aqueous solution with or without BAK across the rabbit sclera was evaluated using a two-chamber Ussing apparatus. To determine the effects of BAK on transscleral delivery of large molecules, 20- and 70-kDa fluorescein isothiocyanate (FITC)-dextran (FD-20 and -70, respectively) aqueous solutions, with or without BAK, were continuously administered to the sclera by an osmotic pump. The intensity of fluorescence in the aqueous humor, vitreous, and retina-choroid was measured by fluorescence spectrophotometry at 1 week after implantation of the pump. The retinal toxicity of BAK was evaluated electrophysiologically and histologically., Results: BAK increased concentrations of BP in the vitreous and retina-choroid compared with the control. BP was not detected in the aqueous humor. In the in vitro study, BAK did not increase the scleral permeability of BP. In the retina-choroid, BAK significantly increased concentrations of FD-20 but did not increase those of FD-70. The addition of BAK did not increase concentrations of FD-20 or -70 in the vitreous. No substantial toxic reactions were observed in the retina in electrophysiological or histologic examinations after the addition of BAK., Conclusions: The results of this study demonstrate that BAK may improve the ocular penetration of a drug in a transscleral drug delivery system without producing toxic reactions.

Purpose: To develop a local drug delivery system that provides therapeutic cyclosporine levels to treat lacrimal gland graft-versus-host disease after allogeneic hematopoietic stem cell transplantation., Methods: Episcleral cyclosporine implants were manufactured with a silicone-based matrix design, and in vitro release rates were determined. Preclinical evaluation included toxicology (clinical examination, serial electroretinography, and histopathology) in normal rabbits and dogs, pharmacokinetics in normal rabbits, and pharmacodynamics in a canine model of aqueous tear deficiency and keratoconjunctivitis sicca., Results: The cyclosporine implants showed sustained release of drug over time with in vitro assays. Histopathology showed normal ocular tissues in both dogs and rabbits 6 months after implantation. The cyclosporine implant produced lacrimal gland drug levels 1 to 2 log units higher than those reported with a variety of topical cyclosporine formulations and oral administration. The cyclosporine implant was effective in a canine model of keratoconjunctivitis sicca, with all animals able to discontinue topical cyclosporine and maintain normal Schirmer scores over a 6-month follow-up., Conclusions: This preclinical evaluation showed that the episcleral cyclosporine implant was safe, delivered potentially therapeutic cyclosporine levels to the lacrimal gland, and showed efficacy in a clinically relevant model of keratoconjunctivitis sicca. The episcleral cyclosporine implant shows promise in reducing the morbidity associated with lacrimal gland graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. In addition, continuous release of cyclosporine in the subconjunctival space with the episcleral implant was an effective means of delivering drug to the ocular surface and may have potential in treating other ocular inflammatory diseases.

Purpose: We hypothesize that remodeling of the scleral extracellular matrix, involving collagen and proteoglycan synthesis and turnover, is a key process involved in ocular growth. Decreased axial elongation is observed following neonatal removal of the crystalline lens in a rhesus monkey model of congenital cataract. We wanted to determine changes in gene expression in the operated and companion eye following lensectomy, especially for extracellular matrix in the sclera., Methods: Between 4 and 7 days of age, infant monkeys underwent surgical removal of the lens from the right eye. Axial lengths of the operated and unmanipulated fellow eyes were measured and when interocular differences of >0.4 mm were achieved, monkeys were sacrificed and RNA was isolated from sclera. In order to determine changes in scleral gene expression in aphakic versus control eyes, we used Clontech's Atlas Gene Array (Human Cancer Array version 1.2) hybridized with total RNA from three monkeys., Results: Atlas Gene Array analysis demonstrated differential expression of several genes in the operated versus the unmanipulated eye. Most notably, there was a statistically significant increase in expression of several extracellular matrix (ECM) genes including: aggrecan, decorin, biglycan, several collagens, and tenascin in the RNA from the sclera of the aphakic eyes when compared to the unmanipulated eyes. Genes for several matrix metalloproteinases (MMPs) showed no significant change following lens removal although there was a trend towards decreased expression. There were also statistically significant changes in the pattern of gene expression in the operated eye relative to the unmanipulated eye for cell adhesion, cell cycle, apoptosis, and cytoskeleton transcripts., Conclusions: Our results suggest that removal of the crystalline lens alters gene expression in the sclera with a prominent upregulation of ECM transcripts. These data support recent evidence that remodeling of the ECM composition of the sclera may be an important regulator of ocular growth.

Ocular penetration of grepafloxacin into several ocular tissues was determined in albino and pigmented rabbits following a single intravitreal administration. After administration, grepafloxacin was detected in all ocular tissues studied in both breeds of rabbits. The superior mean penetration ratios were found in the chorioretina and lens of albino rabbits, and in the chorioretina, iris and lens of pigmented rabbits. A significantly greater penetration of grepafloxacin was found in the chorioretina and iris of the pigmented rabbits than in those of the albino rabbits. As a final conclusion, grepafloxacin detected in different ocular structures could attain therapeutic concentrations against a variety of ocular conditions.

Purpose: To investigate the change in axial eye dimensions resulting from stretching the sclera by acute elevation of intraocular pressure (IOP)., Methods: IOP was increased to 100 mm Hg for 1 hour through an intravitreal cannula, while ocular component dimensions were monitored every 10 minutes with A-scan ultrasound in anesthetized animals (10 chicks and 10 tree shrews). In addition, immunocytochemical detection of alpha-smooth muscle actin (alpha-SMA) using a monoclonal antibody was conducted in the sclera of the tree shrew and the chick., Results: In both species, axial eye length immediately and significantly (P < 0.0001) increased on elevation of IOP to 100 mm Hg: chick to 103.9%, tree shrew to 101.2% (mean percentage of original measured at 15 mm Hg). After 1 hour of maintained pressure, chick eyes showed a further significant increase in axial length (to 108.6%), but axial length of tree shrew eyes decreased (to 100.3%) to the point that it was not significantly different from the original value at 15 mm Hg. Immunocytochemical studies of age-matched tissue demonstrated the presence of alpha-SMA-containing fibroblasts (myofibroblasts) within tree shrew but not chick sclera., Conclusions: Elevation of IOP caused axial elongation of chick eyes, but a consistent reduction in axial length of tree shrew eyes. The presence of myofibroblasts, demonstrated in tree shrew but not chick sclera, suggests that the reduction in axial length of tree shrew eyes may have been caused by activation of a contractile mechanism involving scleral myofibroblasts. Such a mechanism may play a role in the regulation of eye size and refractive development.

We newly prepared a unique one-side-coated insert that releases drug from only uncoated side. The purpose of this study is to determine whether ocular and systemic absorption of ophthalmic drug could be altered by an inserting direction of the insert in rabbit eyes. One-side-coated insert was prepared by attaching a polypropylene tape on the one side of the polymer disc of poly(2-hydroxypropyl methacrylate) (HPM) containing tilisolol as a model ophthalmic drug. The insert was applied in the lower conjunctival cul-de-sac of albino rabbits with the uncoated side facing bulbar conjunctiva/sclera (SC insert) or palpebral conjunctiva (CJ insert). At the adequate intervals, the tear fluid, plasma, aqueous humor, conjunctiva, and sclera were collected and the drug concentrations were determined by an HPLC. A release of tilisolol from the one-side-coated insert was twice slower than from the uncoated insert. Ocular application of the one-side-coated insert produced the constant concentrations of tilisolol in the tear fluid over 180 min. SC insert showed higher drug concentrations in the aqueous humor and sclera, and lower drug concentrations in the plasma and conjunctiva than CJ insert.The one-side-coated insert can alter the ocular and systemic absorption of drug by an inserting direction.

Epithelial membrane protein-2 (EMP2) is a member of the four transmembrane superfamily (TM4SF) and is thought to mediate trafficking of diverse proteins such as alpha6beta1 integrin and MHC class I to lipid raft microdomains. EMP2 has also recently been recognized as a putative tumor suppressor gene in certain model systems. Normally, EMP2 is expressed at discrete locations in the body including high levels in the eye, lung, heart, thyroid, and uterus. Here we examine in detail the subanatomic distribution of EMP2 in murine and human ocular tissue. We observe that EMP2 is localized to epithelial layers of the cornea, ciliary body, and retinal pigmented epithelium-choroid, the stromal layers of the sclera, and the nerve fiber layer of the retina and optic nerve. This distribution is distinct from other TM4SF proteins and may relate to a role in apical membrane recycling.

We determined whether bimatoprost, which has been reported to act via putative prostamide receptors, could be hydrolyzed to its free acid (17-phenyl-PGF(2 alpha)), a potent FP receptor agonist, by human ocular tissue in vitro. We developed a gas chromatography/mass spectrometric method to measure 17-phenyl-PGF(2 alpha) levels at sub-picomolar levels. We then analyzed the amount of 17-phenyl-PGF(2 alpha) present after incubation of 50 microl Lumigan (0.03% bimatoprost) with eye tissue using this assay. We found that cornea, sclera, iris, and ciliary body, all rapidly hydrolyzed bimatoprost to 17-phenyl-PGF(2 alpha) with linear kinetics at a rate of 6.3, 2.0, 2.8, and 1.5 pmol mg tissue(-1) hr(-1), respectively. For cornea, sclera, and ciliary body, this linear rate of hydrolysis continued over a period of at least three hours, while iris-induced hydrolysis did not continue beyond one hour. Our findings suggest that bimatoprost can act as prodrug for FP receptor activation and questions the concept of a "prostamide receptor" agonist.

The objectives of these studies were to determine the amount and distribution of the aminoglycoside antibiotic amikacin delivered to rabbit eyes following transscleral iontophoresis and to determine the inter-study reproducibility of delivery over three identical studies. New Zealand White rabbits (N = 6 per dose group) were treated with a 200-mg/mL amikacin solution at 0, 2, 3 or 4 mA of (+) DC current for 20 minutes. Amikacin concentrations in eye tissues were highest with the 4-mA treatment. Concentrations for all three studies at this current were approximately 5.4, 40, 41, 343, and 92 mcg/g in the vitreous humor, anterior segment, non-treated hemisphere of the sclera, treated hemisphere of the sclera, and retina/choroid, respectively. These values were approximately 27, 50, 40, 10, and 13 fold greater than in the 0-mA control group and are well above the in vitro minimum inhibitory concentrations (MICs) for this drug. Inter-study reproducibility (measured as %CV) depended on the tissue type and treatment group and ranged from 8% for the retina/choroid to 51% for the anterior segment in the 4-mA group. Pretreatment with topical proparacaine hydrochloride local anesthetic did not affect amikacin delivery and total drug delivered was not affected by delivery time for the same total charge administered. Therapeutically relevant amounts of amikacin were delivered into eye tissues in a reproducible and controllable manner.

New methods are presented for simulating steady-state drug concentration in vitreous, choroid, and sclera from an intravitreal device. Clearance by choroidal flow and intraocular pressure (IOP) -induced Darcy hydraulic flow are included. Two methods are proposed for modeling the vasculature using simple one-dimensional models for simulating drug concentration profiles from intravitreal devices. The finite choroid method adds a concentration-dependent sink term to the convective diffusion equation, allowing for a continuous but rapid decrease in concentration throughout the choroid region. The infinitesimal choroid method uses a combination of a simple flux boundary condition and a redefinition of the dependent variable to account for the impact of the vascular drain by a discontinuous drop in concentration across the exterior vitreous boundary. This eliminates the need for a choroidal region, reducing finite element memory requirements, enabling the choroid to be made arbitrarily thin. The impact of permeating fluid induced by IOP on convection was examined, using hydraulic coefficients for ocular tissue recently made available [Xu, J. et al., Pharm. Res. 17:664-669 (2000)], allowing for drug efflux through the outer sclera. Transport becomes diffusion limited at high vascular clearance. Hydraulic flow restricts the range in concentration predicted in the vitreous compared to zero flow. Hydraulic influences for small, rapidly cleared drugs can be neglected.

Purpose: Studies in animal models of refractive development have shown that the development of and recovery from induced myopia is associated with visually-guided changes in scleral glycosaminoglycan synthesis. The present study sought to determine whether differential patterns of scleral glycosaminoglycan synthesis are present in the fibrous scleral layer of the chick during myopia development or recovery, as has previously been reported in the mammalian sclera., Methods: Myopia was induced in young chicks by monocular deprivation of pattern vision over 5 days. Other animals underwent monocular deprivation, then had the occluder removed and were allowed 2 days of recovery. A group of age-matched normal animals served as a control. Newly synthesised glycosaminoglycans in the scleral layers were labelled in vivo, using a [(35)S]-labelled precursor delivered intraperitoneally on the final experimental day. Incorporation of this sulphate into glycosaminoglycans of the fibrous and cartilaginous scleral layers was assessed in proteinase K digests by selective precipitation with alcian blue., Results: Glycosaminoglycan synthesis in the fibrous scleral layers of myopic and recovering eyes was not significantly different to contralateral control eyes (+14 +/- 7%, p = 0.09 and -2 +/- 4%, p = 0.64 respectively). In contrast, glycosaminoglycan synthesis was significantly elevated, relative to controls, in the cartilaginous scleral layer of eyes developing myopia (+63 +/- 18%, p < 0.02), whereas in recovering eyes there was found to be a significant decrease in synthesis in the cartilaginous layer (-40 +/- 6%, p < 0.001)., Conclusions: The results of the current study demonstrate that the fibrous scleral layer of the chick does not display the characteristic differential patterns of glycosaminoglycan synthesis that are found in the mammalian sclera during myopia development and recovery. However, as has previously been reported, the cartilaginous layer of the chick sclera does display differential glycosaminoglycan expression, although the direction of regulation is opposite to that found in the fibrous sclera of mammals.

Purpose: To determine the role in the eye of chondromodulin (ChM)-I, which has been identified in cartilage as an angiogenic inhibitor, the expression and localization and a possible function of ChM-I were investigated., Methods: Expression and localization of ChM-I in rat eyes were examined by RNase protection assay and in situ hybridization and by immunostaining, using an antibody against a synthetic peptide. The effect of recombinant ChM-I on tube morphogenesis of retinal endothelial cells was examined in culture., Results: The rat ChM-I gene was determined to encode the open reading frame of 334 amino acid residues, and ChM-I mRNA was exclusively expressed in cartilage, eye, and cerebellum in rats. ChM-I mRNA expression was evident in the iris-ciliary body, retina, and scleral compartments, but not in other compartments of the eye. In situ hybridization revealed mRNA expression in the ganglion cells, inner nuclear layer cells, and pigment epithelium in the retina and in the nonpigment epithelium of the ciliary body. Immunoreactive ChM-I was present in these cells and also in the vitreous body. Western blot analysis detected an approximately 25-kDa band of ChM-I presumed as a secretory form in the aqueous humor and vitreous body and an approximately 37-kDa band as a precursor form in the retina. Recombinant human ChM-I inhibited tube morphogenesis of human retinal endothelial cells in vitro., Conclusions: These observations indicate a potential role for ChM-I in inhibition of angiogenesis in the rat eye.

Purpose: Myopia can be induced in chickens by having them wear either negative lenses (lens-compensation myopia [LCM]) or diffusers (form-deprivation myopia [FDM]), whereas positive lenses cause lens-compensation hyperopia (LCH). These three conditions were compared with respect to (i) their early time course and (ii) the effect of two manipulations of the lighting., Methods: Longitudinal changes in ocular dimensions and refractive error were measured in chicks maintained under three different conditions: (i) wearing either -15 D lenses or diffusers in a normal light/dark cycle; (ii) wearing either +15 D lenses, -15 D lenses, or diffusers with brief periods of stroboscopic lights at the beginning and end of the dark period; (iii) wearing either +6 D lenses, -6 D lenses, or diffusers with the nights interrupted by brief periods of white light. In addition, scleral and choroidal proteoglycan synthesis was measured in eyes that wore positive lenses, negative lenses, or diffusers for 3 hours followed by different periods of darkness., Results: (i) The time course of the changes in axial length over the first 72 hours was significantly faster in LCM than in FDM. Indeed, the diffusers did not begin to significantly affect the total length of the globe for 3 days, although the vitreous chamber had deepened after 9 hours, because the choroid thinned extremely rapidly (within 1 hour) with either diffusers or negative lenses. (ii) Scleral proteoglycan synthesis was higher in eyes with negative lenses than in those with diffusers at 11 hours, but the reverse was true at 27 hours. (iii) Brief periods of stroboscopic light attenuated FDM more than LCM. (iv) In contrast, interruption of the nights by brief periods of light attenuated LCM more than FDM. (v) Neither lighting manipulation affected LCH. (vi) Choroidal proteoglycan synthesis decreased similarly with 3 hours of wearing either diffusers or negative lenses., Conclusions: Although both negative lenses and diffusers cause similar increases in the rate of ocular elongation, the responses differ in time course and in the effect of manipulations of the daily lighting. The responses to positive lenses differ from both of these.

Purpose: Scleral proteoglycans were characterized from human donor eyes aged 2 months to 94 years to identify age-related changes in the synthesis and/or accumulation of these extracellular matrix components., Methods: Newly synthesized proteoglycans (previously radiolabeled with 35SO4) and total accumulated scleral proteoglycans were extracted with 4 M guanidine hydrochloride and separated by molecular sieve chromatography on a Sepharose CL-4B column. The elution positions of newly synthesized and total accumulated proteoglycans were determined by assaying each fraction for radioactivity and glycosaminoglycans, respectively. Regression analyses were performed on the three major proteoglycan peaks to identify age-related changes in scleral proteoglycan composition. Scleral proteoglycans were further purified by anion-exchange chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses., Results: Human scleral proteoglycans were apparent as three major peaks after chromatography on Sepharose CL-4B. The two faster eluting peaks contained alternative forms of the cartilage proteoglycan, aggrecan, whereas the third peak contained the small proteoglycans biglycan and decorin. The relative percentage of newly synthesized and total accumulated aggrecan increased approximately two- to sixfold from infancy to 94 years. In contrast, the relative percentage of newly synthesized and total accumulated biglycan and decorin decreased by approximately 25%. Chromatography and Western blot results indicated that the absolute amounts of all three proteoglycans significantly increased in concentration within the sclera from birth to the fourth decade. Beyond the fourth decade, decorin and biglycan decreased in all scleral regions and were present in lowest concentrations by the ninth decade. In contrast, aggrecan, which was present in highest concentration in the posterior sclera, was not significantly reduced with increasing age., Conclusions: The age-related changes in scleral proteoglycan composition observed in the present study are likely to contribute to the regional alterations in biomechanical properties of the sclera associated with growth and aging.

Purpose: To study distribution and cellular localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein (TIGR) in the human eye., Methods: A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes., Results: By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm's canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled., Conclusions: Myocilin TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma.

Purpose: To determine the ocular pharmacokinetics of cyclosporin A after topical ophthalmic administration., Methods: Radiolabled cyclosporin A in either a castor oil-in-water emulsion or a corn oil ointment was applied to the eyes of beagle dogs or albino rabbits using the following paradigms: (i) single doses of 0.2% emulsion to rabbits and dogs, (ii) single doses of 0.05%, 0.2%, or 0.4% emulsion to rabbits, (iii) multiple doses of 0.2% emulsion to dogs, (iv) single and multiple doses of 0.2% ointment to rabbits. The distribution of cyclosporin A was determined by measuring the distribution of radioactivity., Results: After a single dose, cyclosporin A was rapidly absorbed into the conjunctiva (Cmax: dogs, 1490 ng/g; rabbits, 1340 ng/g) and cornea (Cmax: dogs, 311 ng/g; rabbits, 955 ng/g). High concentrations (>300 ng/g) could be detected in the cornea up to 96 hours post-dose. Lower concentrations were found in the intraocular tissues, and systemic absorption was minimal. After multiple doses, there was some accumulation in the cornea, lens, lacrimal gland, and iris-cilliary body, but limited accumulation in the conjunctiva and sclera. Ocular tissue concentrations of cyclosporin A increased with increasing dose concentration; proportionally in lacrimal gland and intraocular tissues; less than proportionally in conjunctiva and cornea. The pharmacokinetic profile of the cyclosporin A corn oil ointment was similar to that of the emulsion., Conclusions: Topical ophthalmic cyclosporin A penetrated into extraocular tissues at concentrations adequate for local immunomodulation while penetration into intraocular tissues was much less and absorption into the blood was minimal.

We measured mitomycin C (MMC) concentrations in ocular tissues in rabbits with variable dose (0.1, 0.2, or 0.4 mg) and duration of application time (1, 3, or 5 minutes) of MMC using high-performance liquid chromatography. Mitomycin C concentrations at the administered site after single subconjunctival application of MMC and after irrigation showed significant correlation with dose and duration of time of application. By multiple regression analysis, MMC concentrations (microg/g) at the conjunctiva were described as -6.73 + 67.4 x Dose (mg) + 1.66 x Time (minutes) (R2 0.65); at the sclera, -1.85 + 38.2 x Dose + 0.927 x Time (R2 0.63); at the cornea, -0.727 + 8.44 x Dose (R2 0.46). With a 0.2-mg MMC dose, in all three application times (1, 3, or 5 minutes), MMC concentrations in the conjunctiva at the administered quadrant were three times higher than in the neighboring quadrants and 6 to 7 times higher than in the opposite quadrant. In the sclera, MMC concentrations were 3.5 times higher than in the neighboring sites and over 8 to 9 times higher than in the opposite site. In the cornea, MMC concentrations were 2 to 3 times higher than in the neighboring sites and opposite site. In the iris-ciliary body, MMC concentrations were 0.61 microg/g at the administered site with 0.2 mg for 3-minute application, 2 times higher than in neighboring sites, and 2 times higher than in opposite sites.

Ocular membranes have been characterized by in-situ absorption of the ophthalmic beta-blockers carteolol (hydrophilic) and timolol and befunolol (lipophilic) using a cylindrical cell. After introduction of drug solution into the cell on the cornea, sclera (bulbar conjunctival and scleral layer) or palpebral conjunctiva, the disappearance of the drug from the cell was determined as in-situ absorption. The ophthalmic drugs disappeared from the conjunctival and scleral membranes although disappearance from the cornea was hardly observed. The conjunctival membrane showed the highest permeability. Lipophilic drugs were more permeable than hydrophilic. In-situ apparent permeability coefficients of the ophthalmic drugs through the conjunctiva and sclera correlated with the lipophilicity of drugs. A high drug concentration in the aqueous humor was observed after corneal application. There is a relationship between concentration in the aqueous humor was observed after corneal application. There is a relationship between concentrations of drugs in the aqueous humor and previously reported in-vitro apparent permeability coefficients of the drugs in the cornea. This in-situ method using a cylindrical cell is a useful method of investigating the ocular absorption of ophthalmic drugs.

Purpose: To clarify retinal control on scleral growth in form-deprivation myopia (FDM) in the chick, the authors studied change in transforming growth factor-beta (TGF-beta) in the form-deprived eye and the effect of this growth factor on scleral cell proliferation and axial length., Methods: Change in TGF-beta in FDM in the chick was measured by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblot, and immunohistochemistry. The effect of TGF-beta on [3H]thymidine uptake of scleral chondrocytes was determined by organ culture. Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) were administered to determine the effect of TGF-beta activation on the axial length in normal and FDM eyes., Results: The content of TGF-beta messenger RNA (mRNA) and the active form of TGF-beta protein were reduced in FDM eyes compared with the control specimen. Reduced immunoreactivity of TGF-beta in FDM eyes was found in the photoreceptor layer. The TGF-beta inhibited [3H]thymidine uptake into scleral chondrocytes. In the nondeprived eyes, the vitreous chamber depth and axial length were reduced after uPA treatment, whereas PAI-1 increased them. In the form-deprived eyes, uPA inhibited vitreous depth and axial length elongation, but PAI-1 had no effect., Conclusions: The authors' results suggest that TGF-beta mediates retinal control of ocular growth. Axial elongation in FDM probably is correlated with the reduction of TGF-beta in the retina, retinal pigment epithelium, and choroid. The uPA and PAI-1 treatment controls the activation of TGF-beta and affects axial length.

Studies were conducted in rabbits to determine the ocular distribution and elimination of cyclosporine, with the objective of developing a comprehensive pharmacokinetic model. Following a bolus dose into the anterior chamber, drug levels were measured in the aqueous humor, cornea, iris/ciliary body, lens, sclera, and conjunctiva. Cyclosporine was rapidly eliminated from the aqueous, but drug levels in ocular tissues persisted for in excess of 48 hours, particularly in the cornea and iris/ciliary body. The terminal elimination half life from these tissues was 45 hr and 30 hr, respectively, providing evidence that these tissues could act as a reservoir for the drug. It was found that a compartmental model accurately described the experimental data. A single compartment was used for each of the tissues and fluids sampled, except for the cornea, which was subdivided into two compartments, representing its tissue and aqueous regions.

The elimination of dexamethasone sodium m-sulfobenzoate, DMSB, following intravitreal injection, was measured in rabbit vitreous body under in vivo and in vitro conditions. The rate of elimination in vivo was appreciably greater than that in vitro, indicating that the in vivo data include not only the elimination due to metabolism/degradation in the vitreous humor, but also the elimination through the surrounding tissues such as the posterior aqueous humor, the retina/choroid/sclera membrane, and the lens. A general mathematical model based on Fick's second law of diffusion was developed for describing the pharmacokinetics of the intravitreal injection of DMSB. The model parameters were independently determined from a set of in vitro experiments. The in vivo data of elimination of DMSB following intravitreal injection agreed with the profiles calculated from the mathematical model, together with the model parameters determined from the in vitro experiments. The present in vivo/in vitro correlation, with the help of computer simulation, can be used for optimizing the therapeutic systems of intravitreal drug delivery.