Purpose

To investigate the effects of chronic oxidative stress on lysosomal function in trabecular meshwork (TM) cells.

Methods

Confluent cultures of porcine TM cells were grown for 2 weeks in physiological (5% O2) or hyperoxic conditions (40% O2) in the presence or absence of the protease inhibitor leupeptin (10 μM). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular reactive oxygen species (ROS; H2DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (Mitotracker Red; Invitrogen-Molecular Probes, Eugene, OR), lysosomal content (acridine orange and Lysotracker Red [Invitrogen-Molecular Probes]), autophagic vacuole content (MDC), SA-β-galactosidase (FDG), and cathepsin activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western blot analysis. Ultrastructural analysis was performed by transmission electron microscopy.

Results

Prolonged exposure of porcine TM cells to a hyperoxic environment led to an increase in ROS production and oxidized material. Electron micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded lysosomes. Augmented lysosomal and autophagic vacuole content was confirmed with specific fluorophores. The mRNA and protein levels of several cathepsins were upregulated with oxidative stress. This up-regulated expression did not correlate with increased lysosomal activity.

Conclusions

The results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment, leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.

Purpose

To test AAV-mediated gene therapy in the rd10 mouse, a natural model of recessive RP caused by mutation of the β-subunit of rod photoreceptor cGMP phosphodiesterase.

Methods

One eye of a cohort of rd10 mice kept in a dark environment was subretinally injected at postnatal day (P) 14 with 1 μL AAV5-smCBA-PDEβ. The contralateral eye was not injected. The animals were then maintained for 2 weeks in the dark before they were moved to a normal 12-hour light/12-hour dark cycling light environment for visually guided behavioral training. Three weeks after injection, treated rd10 mice were examined by scotopic and photopic electroretinography and then killed for biochemical and morphologic examination.

Results

Substantial scotopic ERG signals were maintained in treated rd10 eyes, whereas untreated eyes in the same animals showed minimal signals. Treated eyes showed photopic ERG b-wave amplitudes similar to those of the normal eyes; in untreated partner eyes, only half the normal amplitudes remained. Strong PDEβ expression was observed in photoreceptor outer segments only in treated eyes. Light microscopy showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Electron microscopy showed good outer segment preservation only in treated eyes. A visually guided water maze behavioral test under dim light showed significantly improved performance in one eye–treated rd10 mice compared with untreated mice.

Conclusions

These data demonstrate that P14 administration of AAV5-smCBA-PDEβ can prevent retinal degeneration in rd10 mice, as reflected by significant structural, biochemical, electrophysiological, and behavioral preservation/restoration. These results serve as a baseline for studying long-term retinal rescue in rd10 mice.

Purpose

To evaluate production and activity of metalloproteinase (MMP)-9 on the ocular surface of patients with dysfunctional tear syndrome (DTS) and determine any correlation between MMP-9 activity and clinical parameters.

Methods

Forty-six patients with newly diagnosed DTS and 18 control subjects were recruited. Complete ocular surface examinations were performed. Tear MMP-9 activity was assessed with an MMP-9 activity assay in 1 μL of unstimulated tear fluid. Using conjunctival epithelial cells from 19 patients with DTS and 16 controls, levels of MMP-9 and its regulating cytokine mRNA transcripts were evaluated by semiquantitative real-time PCR.

Results

Each of four DTS severity–based groups had significantly higher mean MMP-9 activities than did the control group, which was 8.39 ± 4.70 ng/mL. The DTS4 group had the highest MMP-9 activity (381.24 ± 142.83 ng/mL), for which the mean was significantly higher than that of other DTS groups. In addition, patients with DTS had significantly higher levels of IL-1β, IL-6, TNF-α, and TGF-β1 mRNA transcripts in their conjunctival epithelia than did the control subjects. Tear MMP-9 activities showed significant correlation with symptom severity scores, decreased low-contrast visual acuity, fluorescein tear break-up time, corneal and conjunctival fluorescein staining, topographic surface regularity index (SRI), and percentage area of abnormal superficial corneal epithelia by confocal microscopy.

Conclusions

Tear MMP-9 activity was significantly higher in patients with DTS. This activity was associated with increased mRNA expression of MMP-9 and its regulating genes and correlated strongly with clinical parameters. MMP-9 appears to be a potentially useful biomarker for diagnosing, classifying, and monitoring DTS.

Purpose

Elevated hydrostatic pressure induces retinal ganglion cell (RGC) apoptosis in culture. The authors investigated whether the transient receptor potential vanilloid 1 (TRPV1) channel, which contributes to pressure sensing and Ca2+-dependent cell death in other systems, also contributes to pressure-induced RGC death and whether this contribution involves Ca2+.

Methods

trpv1 mRNA expression in RGCs was probed with the use of PCR and TRPV1 protein localization through immunocytochemistry. Subunit-specific antagonism (iodo-resiniferatoxin) and agonism (capsaicin) were used to probe how TRPV1 activation affects the survival of isolated RGCs at ambient and elevated hydrostatic pressure (+70 mm Hg). Finally, for RGCs under pressure, the authors tested whether EGTA chelation of Ca2+ improves survival and whether, with the Ca2+ dye Fluo-4 AM, TRPV1 contributes to increased intracellular Ca2+.

Results

RGCs express trpv1 mRNA, with robust TRPV1 protein localization to the cell body and axon. For isolated RGCs under pressure, TRPV1 antagonism increased cell density and reduced apoptosis to ambient levels (P ≤ 0.05), whereas for RGCs at ambient pressure, TRPV1 agonism reduced density and increased apoptosis to levels for elevated pressure (P ≤ 0.01). Chelation of extracellular Ca2+ reduced RGC apoptosis at elevated pressure by nearly twofold (P ≤ 0.01). Exposure to elevated hydrostatic pressure induced a fourfold increase in RGC intracellular Ca2+ that was reduced by half with TRPV1 antagonism. Finally, in the DBA/2 mouse model of glaucoma, levels of TRPV1 in RGCs increased with elevated IOP.

Conclusions

RGC apoptosis induced by elevated hydrostatic pressure arises substantially through TRPV1, likely through the influx of extracellular Ca2+.

Purpose

To quantify the forces necessary to change the shape and optical power of human and monkey lenses.

Methods

Cynomolgus monkey (n = 48; age: 3.8–11 years), rhesus monkey (n = 35; age: 0.7–17 years) and human (n = 20, age 8–70 years) eyes obtained postmortem, including the lens, capsule, zonules, ciliary body, and sclera were mounted in an optomechanical lens-stretching system. Starting at zero load, the lenses were symmetrically stretched in a stepwise fashion in 0.25- or 0.5-mm steps. The load, lens diameter, inner ciliary body diameter, and lens power were measured at each step and the diameter- and power-load responses were quantified.

Results

The diameter- and power-load responses were found to be linear in the physiologically relevant range of stretching. The average change in cynomolgus, rhesus, and human lens diameter, respectively, was 0.094, 0.109, and 0.069 mm/g in young lenses, and 0.069, 0.067, and 0.036, mm/g in older lenses. For the same lenses, the average change in lens power was −3.73, −2.83, and −1.22 D/g in young lenses and −2.46, − 2.16, and −0.49 D/g in older lenses.

Conclusions

The force necessary to change the lens diameter and lens power increases with age in human and monkey lenses. The results agree with the Helmholtz theory of accommodation and with presbyopia theories that predict that the force required to disaccommodate the lens increases with age.

Purpose

Proliferative vitreoretinopathy (PVR) is the primary cause of failure of retinal reattachment surgery. Growth factors such as platelet-derived growth factor (PDGF) are strongly associated with PVR. Of the five PDGF family members, PDGF-C predominates in the vitreous of experimental and clinical PVR. PDGF-C is secreted as a latent protein that requires proteolytic processing for activation. Although tissue plasminogen activator (tPA) is primarily responsible for processing PDGF-C in cultured cells, it constitutes a minority of the processing activity in the vitreous of experimental animals and in patients with PVR. Identifying the major PDGF-C processing protease was the purpose of this study.

Methods

The presence of serum proteins in the vitreous was detected by Coomassie blue staining and Western blotting. PDGF-C processing activity was detected in an in vitro processing assay using either native or recombinant PDGF-C as the substrate. Plasmin activity was blocked using α2-plasmin inhibitor. Phosphorylation of the PDGF receptor (PDGFR) was monitored by antiphosphotyrosine Western blotting. Vitreous specimens were collected from experimental rabbits or from patients undergoing vitrectomy to repair retinal detachment or for other reasons.

Results

A number of prominent serum proteins (albumin and IgG) were detected in the vitreous of all patients undergoing retinal surgery. The level of these proteins markedly increased in the vitreous of rabbits as they developed PVR. These observations suggested that serum-borne proteases are also likely to be present in the vitreous. Indeed, plasmin (a protease capable of processing PDGF-C) was present in the vitreous from PVR rabbits and retinal surgery patients. Plasmin was dramatically more effective than tPA in processing PDGF-C in an in vitro assay. Blocking plasmin activity eliminated most of the processing activity in the vitreous of patients and rabbits with PVR.

Conclusions

Plasmin was the major PDGF-C processing protease in the vitreous of PVR rabbits and patients undergoing retinal surgery. Blocking plasmin prevented the generation of active PDGF-C, which is the major PDGF isoform relevant for PVR. These observations are the first report of an in vivo protease responsible for processing PDGF-C. In addition, plasmin was identified as a novel therapeutic target for patients with PVR.

Purpose

To determine the repeatability of real-time optical coherence tomography (OCT) measurements of tear film thickness (TFT) and variables of tear film menisci.

Methods

Forty eyes were imaged with a custom-built, real-time OCT to obtain heights, curvatures, and cross-sectional areas of upper and lower tear menisci simultaneously. The central TFT was indirectly determined as the difference between the combined thickness of the central cornea and tear film and the true corneal thickness obtained after instillation of artificial tears. Dynamic tear distribution was determined by OCT imaging immediately and 5, 20, 40, and 60 minutes after tear instillation. Measurements taken after two blinks of one eye at each visit were repeated on the next day. Measurements from the companion eye were made on separate days.

Results

There were no significant differences between the two measurements of each variable made on consecutive days. At baseline, upper tear meniscus variables were strongly correlated with the comparable lower meniscus variables. However, there were no significant correlations between TFT and any tear meniscus variable. Immediately after instillation of artificial tears, all measured variables increased significantly. TFT, upper and lower menisci heights, and upper meniscus area remained elevated for at least 5 minutes. In addition there were significant correlations between TFT and the lower tear meniscus height and area.

Conclusions

The custom-built OCT showed good repeatability and holds promise in measuring the dynamic distribution of artificial tears on the ocular surface.

Purpose

To compare drivers and non-drivers, and describe the specific concerns of drivers, among individuals with glaucoma.

Methods

607 newly-diagnosed glaucoma patients from 14 clinical centers of the Collaborative Initial Glaucoma Treatment Study were randomly assigned to initial medicine or surgery and followed every six months for < 5 years. Driving status (drivers vs. non-drivers) as well as patient-reported visual function was determined by the Visual Activities Questionnaire and the National Eye Institute Visual Function Questionnaire. Clinical evaluation included visual field mean deviation (MD) and visual acuity. Statistical comparisons were made using t, Chi-square, and exact tests, regression, and Rasch analyses.

Results

Drivers were more likely than non-drivers to be male, white, married, employed, and have more education, higher income, and fewer co-morbidities. Over 50% of drivers reported at least “some” difficulty performing tasks involving glare, whereas 22% reported at least “some” difficulty with tasks requiring peripheral vision. At 54 months, drivers with moderate/severe bilateral visual field loss (VFL) reported greater difficulty with night driving and tasks involving visual search and visual processing speed than drivers with less bilateral VFL (all p-values <0.05). While those who remained drivers over follow-up had better MD in both eyes than those who became non-drivers due to eyesight, a number of drivers had marked VFL.

Conclusion

Inquiring about specific difficulties with tasks related to glare, visual processing speed, visual search and peripheral vision in driving, especially among patients with substantial bilateral VF damage, will enable physicians to more effectively counsel patients regarding driving.

Purpose

Recent investigations have revealed that populations of macrophages and dendritic cells (DCs) are present in the stroma and epithelium of the cornea, although the precise phenotype and distribution are still controversial. CX3CR1, the sole receptor for the chemokine fractalkine, is expressed by these monocyte-derived cells. Transgenic CX3CR1GFP mice, in which either one (heterozygous) or both (homozygous) copies of the CX3CR1 gene were replaced by enhanced green fluorescent protein (eGFP), were used to characterize monocyte-derived cells in the mouse cornea and to determine whether the expression of this receptor influences the recruitment of these cells into the normal cornea.

Methods

Wholemount corneas were immunostained with anti-leukocyte antibodies to the phenotypic markers major histocompatibility complex (MHC) class II, CD169, CD68, CD11b, and CD45 and analyzed by epifluorescence and confocal microscopy. The density of intraepithelial MHC class II+ cells was quantified in wild-type, CX3CR1+/GFP heterozygous, CX3CR1GFP/GFP homozygous, and CX3CR1-knockout mice.

Results

There was a significant reduction in the number of MHC class II+ cells (putative DCs) in the corneal epithelium of CX3CR1-deficient mice (P < 0.009) compared with wild-type mice, and the few cells that were present did not possess classic dendriform morphology. No GFP+ MHC class II– cells were noted in the epithelium. Dual immunostaining of corneas in both heterozygous and homozygous (CX3CR1-deficient) mice revealed GFP+ cells with a more pleomorphic morphology throughout the entire corneal stroma that were CD11b+ CD169+, and had variable degrees of expression of CD68 and MHC class II. The immunophenotype and morphology of these intrastromal cells is strongly indicative of a macrophage phenotype.

Conclusions

This study has identified a role for CX3CR1 in the normal recruitment of MHC class II+ putative DCs into the corneal epithelium and establishes a model for investigating monocyte-derived cells and fractalkine/CX3CR1 interactions during corneal disease.

PURPOSE

To evaluate the birefringence properties of eyes with age-related macular degeneration (AMD). To compare the information from two techniques—scanning laser polarimetry (GDx) and polarization-sensitive spectral-domain optical coherence tomography (OCT)—and investigate how they complement each other.

METHODS

The authors prospectively examined the eyes of two healthy subjects and 13 patients with exudative AMD. Using scanning laser polarimetry, they computed phase-retardation maps, average reflectance images, and depolarized light images. To obtain polarimetry information with improved axial resolution, they developed a fiber-based, polarization-sensitive, spectral-domain OCT system and measured the phase retardation associated with birefringence in the same eyes.

RESULTS

Both GDx and polarization-sensitive spectral-domain optical coherence tomography detected abnormal birefringence at the locus of exudative lesions. Polarization-sensitive, spectral-domain OCT showed that in the old lesions with fibrosis, phase-retardation values were significantly larger than in the new lesions (P = 0.020). Increased scattered light and altered polarization scramble were associated with portions of the lesions.

CONCLUSIONS

GDx and polarization-sensitive spectral-domain OCT are complementary in probing birefringence properties in exudative AMD. Polarimetry findings in exudative AMD emphasized different features and were related to the progression of the disease, potentially providing a noninvasive tool for microstructure in exudative AMD.

Purpose

The goal of these experiments was to identify the neurotransmitter in centrifugal axons of the macaque retina.

Methods

Macaca mulatta, retinas and optic nerves were fixed overnight in carbodiimide and labeled with an antiserum to histamine with the use of an immunofluorescence technique.

Results

Several large histamine-immunoreactive axons ran from the optic nerve head to the peripheral retina, where they branched extensively and terminated in the inner plexiform layer, occasionally alongside retinal blood vessels. Other axons that emerged from the optic nerve head ran in the optic fiber layer to the central retina, circled the fovea, and then returned to the optic disc. These may be the source of histamine-immunoreactive axons that have been observed in central visual areas. No labeled cell bodies were present in the retina. Because perikarya in die posterior hypothalamus are the only known source of histamine in the primate central nervous system and because neurons there can be retrogradely labeled from the cut optic nerve, the histamine-immunoreactive axons must have originated there.

Conclusions

Centrifugal axons in the macaque retina are part of the system of axons containing histamine that originate in the hypothalamus and project throughout the brain. Because the activity of these neurons is highest during the morning, histamine might play a role in preparing the retina to operate in daylight. The contacts of histamine-immunoreactive axons with blood vessels suggest that histamine may also play a role in regulating the retinal microvasculature.

Purpose

The goal was to understand the functions of retinopetal axons containing histamine. In prior work, type 3 histamine receptors (HR3) have been localized to the tips of ON bipolar cell dendrites in macaque retinas. Voltage-gated potassium channels have also been localized to bipolar cell dendrites, and the hypothesis tested in the present study was that these are modulated by histamine.

Methods

Whole-cell recordings of potassium currents were made from bipolar cells in slice preparations of macaque retina. In voltage-clamp mode, the cells were held at −60 mV and stepped to values from −60 to 80 mV. Recordings of the membrane potential were also made in current-clamp mode. Histamine, the HR3 agonist (R) α-methylhistamine (RAMH), tetraethyl ammonium (TEA), and 4-aminopyridine (4-AP) were applied in the superfusate.

Results

Histamine produced a dose-dependent increase in potassium currents in a subset of bipolar cells. At 5 μM, histamine increased the currents by 15% or more in the ON bipolar cells but not in the OFF bipolar cells. RAMH at 5 μM increased the amplitude of the potassium currents in the ON bipolar cells. In 10 mM TEA, potassium currents were reduced in all the bipolar cells, and there was no effect of histamine. Histamine hyperpolarized the resting membrane potential of the ON bipolar cells by 5 mV.

Conclusions

By enhancing potassium currents in the ON bipolar cells, histamine is expected to reduce the amplitude of the light responses and limit their duration. The hyperpolarization of the resting membrane potential would also reduce neurotransmitter release at their output synapses.

Purpose

To characterize the effects of diabetes on the expression of histidine decarboxylase mRNA and on the morphology of the histaminergic centrifugal axons in the rat retina.

Methods

Rats were made diabetic by streptozotocin. After 3 months, retinal histidine decarboxylase expression was analyzed by in situ hybridization in radial sections. Flatmount retinas from a second group of rats were labeled with an antiserum to histamine or an antibody to phosphorylated neurofilament protein.

Results

Histidine decarboxylase mRNA was expressed in cells in the inner and outer nuclear layers of diabetic retinas, but not in normal retinas. However, immunoreactive (IR) histamine was not localized to perikarya in either the normal or the diabetic retinas. Instead, a population of centrifugal axons was labeled. These axons emerged from the optic disc and had varicose terminal branches in the inner plexiform layer (IPL) of the peripheral retina. Some branches ended on large retinal blood vessels and others in dense clusters in the IPL. In rats with streptozotocin-induced diabetes, the centrifugal axon terminals developed many large swellings that contained neurofilament immunoreactivity; these swellings were rare in normal retinas.

Conclusions

Diabetes perturbs the retinal histaminergic system, causing increases in histidine decarboxylase mRNA expression in neurons or glia and abnormal focal swellings on the centrifugal axons.

Purpose

Too investigate asymmetry in eyelid movements with blinking, the stability of the asymmetry, and its modifiability in normal humans.

Methods

Differences in the start time and amplitude between the two eyelids were assessed for voluntary blinks and reflex blinks evoked by supraorbital trigeminal nerve stimulation. These variables were also measured before and up to 18 months after 2 hours of unilateral upper lid restraint.

Results

With voluntary blinks, one eyelid consistently began to close earlier and made a larger eyelid movement than the other eyelid. Stimulation of the supraorbital branch of the trigeminal nerve evoked relatively larger amplitude blinks in one eyelid that correlated with the asymmetries of voluntary blinks. There was a continuum of eyelid asymmetry across all subjects that was stable and independent of other biological asymmetries, such as handedness. Briefly reducing eyelid mobility created a long-lasting change in eyelid asymmetry with blinking.

Conclusions

Eyelid asymmetry results from differences in the excitability of motoneurons in the left and right facial motor nuclei and does not appear to involve asymmetries in cortical inputs to the brain stem. Because adaptive processes modify the motoneuron excitability that creates eyelid asymmetry, these processes may underlie changes in blinking associated with facial palsy and may play a role in the development of disorders that affect one side of the face, such as hemifacial spasm.

Purpose

To determine the influence of eye pigmentation on transscleral retinal delivery of celecoxib.

Methods

Melanin content in ocular tissues of both the strains was determined by sodium hydroxide solubilization method. The affinity of celecoxib to synthetic and natural melanin was estimated by co-incubating celecoxib and melanin in isotonic phosphate-buffered saline. The binding affinity (k) and the maximum binding (rmax) for celecoxib to both natural and synthetic melanin were estimated. Suspension of celecoxib (3 mg/rat) was injected periocularly into one eye of Sprague-Dawley (SD, albino) and Brown Norway (BN, pigmented) rats. The animals were euthanatized at the end of 0.25, 0.5, 1, 2, 3, 4, 8, or 12 hours after the drug was administered, and celecoxib levels in ocular tissues (sclera, choroid-RPE, retina, vitreous, lens, and cornea) were estimated with an HPLC assay. In addition, celecoxib-poly(lactide) microparticles (750 μg drug/rat) were administered periocularly in SD and BN rats, and celecoxib levels in these eye tissues were assessed on day 8, to determine the effectiveness of the sustained release system.

Results

The rmax and k for celecoxib’s binding to natural melanin were (3.92 ± 0.06) × 10−7 moles/mg of melanin and (0.08 ± 0.01) × 106 M−1, respectively. The affinity and the extent of celecoxib’s binding to natural melanin were not significantly different from those observed with synthetic melanin. The concentrations of melanin in choroid-RPE, sclera, and retina of BN rats were 200 ± 30, 12 ± 4, and 3 ± 0.2 μg/mg tissue, respectively. Melanin was not detectable in the vitreous, lens, and cornea of BN rats. In SD rats, melanin was not detected in all tissues assessed except in the choroid-RPE, wherein melanin-like activity was 100-fold less than in BN rats. The area under the curve (AUC) for tissue concentration versus time profiles for animals administered with celecoxib suspension was not significantly different between the two strains for sclera, cornea, and lens. However, the retinal (P = 0.001) and vitreal (P = 0.001) AUCs of celecoxib in the treated eyes were approximately 1.5-fold higher in SD rats than in BN rats. Further, the choroid-RPE AUC in the treated and untreated eyes, respectively, were 1.5-fold (P = 0.001) and 2-fold (P = 0.0001) higher in BN rats than in SD rats. With celecoxib-poly(lactide) microparticles, choroid-RPE, retina, and vitreous concentrations on day 8 exhibited similar trends in differences between the two strains, with the differences being greater than those recorded for the celecoxib suspension.

Conclusions

Transscleral retinal and vitreal drug delivery of lipophilic celecoxib is significantly lower in pigmented rats than in albino rats. This difference may be attributable to significant binding of celecoxib to melanin and its accumulation/retention in the melanin-rich choroid-RPE of pigmented rats. The hindrance of retinal and vitreal drug delivery by the choroid-RPE in pigmented rats is also true of sustained-release microparticle systems.

Purpose

To determine the influence of the choroid–Bruch’s layer and solute lipophilicity on in vitro transscleral drug permeability in bovine and porcine eyes.

Methods

The in vitro permeability of two VEGF inhibitory drugs, budesonide and celecoxib, which are lipophilic and neutral at physiologic pH, and of three marker solutes, 3H-mannitol (hydrophilic, neutral), sodium fluorescein (hydrophilic, anionic), and rhodamine 6G (lipophilic, cationic), were determined across freshly excised scleras, with or without the underlying choroid–Bruch’s layer. Select studies were performed using porcine sclera with and without choroid–Bruch’s layer. Neural retina was removed by exposure of the eyecup to isotonic buffer and wherever required, the retinal pigment epithelial (RPE) layer of the preparation was disrupted and removed by exposure to hypertonic buffer. Because of the poor solubility of celecoxib and budesonide, permeability studies were conducted with 5% wt/vol of hydroxypropyl-β-cyclodextrin (HPβCD). For other solutes, permeability studies were conducted, with and without HPβCD. Partitioning of the solutes into bovine sclera and choroid–Bruch’s layer was also determined.

Results

The calculated log (distribution coefficient) values were −2.89, −0.68, 2.18, 3.12, and 4.02 for mannitol, sodium fluorescein, budesonide, celecoxib, and rhodamine 6G, respectively. Removal of RPE was confirmed by transmission electron microscopy and differences in the transport of mannitol. The order of the permeability coefficients (Papp) across sclera and sclera–choroid–Bruch’s layers in bovine and porcine models was 3H-mannitol > fluorescein > budesonide > celecoxib > rhodamine 6G, with HPβCD, and 3H-mannitol > fluorescein > rhodamine 6G, without HPβCD. The presence of choroid–Bruch’s layer reduced the bovine scleral permeability by 2-, 8-, 16-, 36-, and 50-fold and porcine tissue permeability by 2-, 7-, 15-, 33-, and 40-fold, respectively, for mannitol, sodium fluorescein, budesonide, celecoxib, and rhodamine 6G. The partition coefficients measured in bovine tissues correlated positively with the log (distribution coefficient) and exhibited a trend opposite that of transport. The partition coefficient ratio of bovine choroid–Bruch’s layer to sclera was ~1, 1.5, 1.7, 2, and 3.5, respectively, for the solutes, as listed earlier.

Conclusions

The choroid–Bruch’s layer is a more significant barrier to drug transport than is sclera. It hinders the transport of lipophilic solutes, especially a cationic solute, more than hydrophilic solutes and in a more dramatic way than does sclera. The reduction in transport across this layer directly correlates with solute binding to the tissue. Understanding the permeability properties of sclera and underlying layers would be beneficial in designing better drugs for transscleral delivery.

Purpose

To determine whether celecoxib inhibits VEGF secretion from ARPE-19 cells and to investigate further the safety and effectiveness of periocular celecoxib-poly (lactide-co-glycolide; PLGA) microparticles in inhibiting elevations in retinal PGE2, VEGF, and blood–tissue barrier leakage at the end of 60 days in a streptozotocin diabetic rat model.

Methods

VEGF mRNA and protein expression in ARPE-19 cells was evaluated in the presence of 0 to 10 μM celecoxib, and cytotoxicity of celecoxib on ARPE-19 and RF6A cells was evaluated over a 0- to 100-μM concentration range. Celecoxib-PLGA microparticles were prepared by a modified solvent evaporation technique, sterilized by 25 kGy of γ-irradiation, and characterized for size, ζ potential, drug loading, and in vitro release. Normal and streptozotocin-diabetic male Sprague-Dawley rats were divided into five groups: normal, diabetic, diabetic+placebo, normal+celecoxib, and diabetic+celecoxib. Phosphate-buffered saline (PBS) containing celecoxib-PLGA microparticles, placebo PLGA microparticles, or plain PBS in one eye was injected into the posterior subconjunctival (periocular) space in rats under anesthesia. Sixty days after administration, the animals were killed, and retinal PGE2 secretion, VEGF protein, and blood–retinal barrier leakage were estimated. Blood cell counts, blood chemistry and histology were used to assess the safety of the microparticulate system.

Results

Celecoxib (up to 25 μM) did not cause significant cytotoxicity in ARPE-19 or RF6A cells. Nanomolar concentrations of celecoxib reduced VEGF mRNA and VEGF protein secretion. Celecoxib-PLGA microparticles (diameter: 1140 ± 15 nm), containing 14.93% ± 0.21% of celecoxib sustained in vitro drug release and in vivo drug levels in the retina for 60 days. Diabetes elevated PGE2 secretion, VEGF protein, the vitreous-plasma protein ratio, and blood–retinal barrier leakage by 3-, 1.7-, 3.1-, and 2.7-fold, and celecoxib-PLGA microparticles significantly reduced these elevations by 40%, 50%, 40%, and 50%, respectively. Neither the placebo-treated eyes nor the contralateral eyes in celecoxib-PLGA microparticle-treated rats showed significant effects. Celecoxib-PLGA or placebo-PLGA particles had no effect on the body weight or blood sugar level of rats. The celecoxib-PLGA microparticles did not cause any changes in blood cell counts or chemistry and caused no histopathological damage to the retina or periocular tissues.

Conclusions

Nanomolar concentrations of celecoxib can inhibit VEGF mRNA and protein expression from ARPE-19 cells. Periocular celecoxib microparticles are useful sustained drug delivery systems for inhibiting diabetes-induced elevations in PGE2, VEGF, and blood–retinal barrier leakage. The periocular celecoxib-PLGA microparticles are safe and do not cause any damage to the retina.

Purpose

To develop pharmacokinetics models to describe the disposition of small lipophilic molecules in the cornea and retina after periocular (subconjunctival or posterior subconjunctival) administration.

Methods

Compartmental pharmacokinetics analysis was performed on the corneal and retinal data obtained after periocular administration of 3 mg of celecoxib (a selective COX-2 inhibitor) to Brown Norway (BN) rats. Berkeley Madonna, a differential and difference equation–based modeling software, was used for the pharmacokinetics modeling. The data were fit to different compartment models with first-order input and disposition, and the best fit was selected on the basis of coefficient of regression and Akaike information criteria (AIC). The models were validated by using the celecoxib data from a prior study in Sprague-Dawley (SD) rats. The corneal model was also fit to the corneal data for prednisolone at a dose of 2.61 mg in albino rabbits, and the model was validated at two other doses of prednisolone (0.261 and 26.1 mg) in these rabbits. Model simulations were performed with the finalized model to understand the effect of formulation on corneal and retinal pharmacokinetics after periocular administration.

Results

Celecoxib kinetics in the BN rat cornea can be described by a two-compartment (periocular space and cornea, with a dissolution step for periocular formulation) model, with parallel elimination from the cornea and the periocular space. The inclusion of a distribution compartment or a dissolution step for celecoxib suspension did not lead to an overall improvement in the corneal data fit compared with the two-compartment model. The more important parameter for enhanced fit and explaining the apparent lack of an increase phase in the corneal levels is the inclusion of the initial leak-back of the dose from the periocular space into the precorneal area. The predicted celecoxib concentrations from this model also showed very good correlation (r = 0.99) with the observed values in the SD rat corneas. Similar pharmacokinetics models explain drug delivery to the cornea in rat and rabbit animal models. Retinal pharmacokinetics after periocular drug administration can be explained with a four-compartment (periocular space, choroid-containing transfer compartment, retina, and distribution compartment) model with elimination from the periocular space, retina, and choroid compartment. Inclusion of a dissolution–release step before the drug is available for absorption or elimination better explains retinal tmax. Good fits were obtained in both the BN (r = 0.99) and SD (r = 0.99) rats for retinal celecoxib using the same model; however, the parameter estimates differed.

Conclusions

Corneal and retinal pharmacokinetics of small lipophilic molecules after periocular administration can be described by compartment models. The modeling analysis shows that (1) leak-back from the site of administration most likely contributes to the apparent lack of an increase phase in corneal concentrations; (2) elimination via the conjunctival or periocular blood and lymphatic systems contributes significantly to drug clearance after periocular injection; (3) corneal pharmacokinetics of small lipophilic molecules can be explained by using similar models in rats and rabbits; and (4) although there are differences in some retinal pharmacokinetics parameters between the pigmented and nonpigmented rats, the physiological basis of these differences has yet to be ascertained.

Purpose

Intracameral (anterior chamber) injection of antigen inhibits the development of delayed-type hypersensitivity, a phenomenon known as anterior chamber-associated immune deviation (ACAID). The authors investigated the effect of intracameral injection of interphotoreceptor retinoid-binding protein (IRPB) peptides on the development of IFN-γ+ and IL-17+ pathogenic T cells.

Methods

A uveitogenic (IRBP1–20) or nonuveitogenic (IRBP161–180) peptide was injected into the anterior chamber (AC) of B6 mice. Seven days later, the mice were primed with a pathogenic dose of IRBP1–20 in adjuvant. Thirteen days later, the pathogenic activity of the T cells isolated from the spleens of treated and untreated mice were compared, and the numbers of Th1 and Th17 T cells were assessed by intracellular staining. Regulatory T-cell activity was assessed by antibody staining and functional assays. The authors also compared the effect of inhibition on EAU of ocular injection to various sites, including the AC, the vitreous cavity, and the subretinal space.

Results

Intraocular injection of the uveitogenic peptide (IRBP1–20), but not the nonuveitogenic peptide (IRBP161–180), inhibited the generation of IFN-γ+ and IL-17+ uveitogenic T cells and the development of experimental autoimmune uveitis (EAU). AC administration of IRBP1–20, but not IRBP161–180, significantly decreased the number of circulating γδ T cells after subsequent systemic immunization with IRBP1–20. Absence of the γδ T-cell population prohibited the development of ACAID.

Conclusions

Injection of a uveitogenic peptide into the AC inhibited the development of EAU by regulation of Th1 and Th17 IRBP-specific T cells. The circulating γδ T-cell population was reduced and was associated with decreased activation of IL-17+ uveitogenic T cells.

Purpose

To determine whether Toll-like receptor (TLR) ligands regulate the activation of retinal astrocytes (RACs) and the possible role of RACs in the polarization of interphotoreceptor retinoid-binding protein (IRBP)-specific T cells.

Methods

TLR expression on RACs isolated from C57BL/6 mice was examined using real-time PCR and flow cytometry. The ability of RACs before or after treatment with TLR ligands to interact with T cells was assessed by measuring major histocompatibility complex class II and costimulatory molecule expression and cytokine production. The stimulatory effect of RACs, with or without TLR stimulation, on responder IRBP-specific T cells was examined by T-cell proliferation, cytokine production, and disease-inducing ability.

Results

Cultured mouse RACs expressed TLR2, TLR3, and TLR4. Different TLR ligands had distinct stimulatory effects on RACs. PolyI:C (a TLR3 ligand) had the greatest effect in stimulating RACs to acquire antigen-presentation function, whereas BLP (a TLR2 ligand) had the lowest effect. TLR3 ligation increased the expression of MHC and costimulatory molecules and induced the production of IL-6, IL-12, and IL-23 by RACs. IRBP-specific T cells activated by polyI:C-treated RACs expanded vigorously, produced significant amounts of IFN-γ and IL-17, and induced experimental autoimmune uveitis when injected into naive mice.

Conclusions

The stimulatory effect of RACs on autoreactive T cells is regulated by TLR ligands. TLR3 had a marked effect on the ability of RACs to promote the activation of Th1 and Th17 IRBP-specific T cells. Thus, exposure to microbial antigen(s) may alter susceptibility to autoimmune uveitis by promoting the activation of autoreactive T cells.

Purpose

To examine the normal murine corneal stroma for the presence of bone marrow–derived leukocytes.

Methods

Wholemounts of paraformaldehyde-fixed corneal stroma from normal mice at 5 to 16 weeks of age were examined in single- and double-color immunomorphologic studies performed with confocal microscopy. The phenotype, morphology, distribution, and density of immunopositive cells were determined.

Results

Numerous CD45+ cells with pleomorphic and dendriform morphology were found within the pericentral and central region of the corneal stroma (200–300 cells/mm2). Dual-color immunostaining demonstrated that 100% of the CD45+ cells coexpressed CD11b and 50% coexpressed F4/80. Approximately 30% of the total cells and 50% of the F4/80+ cells coexpressed major histocompatibility complex (MHC) class II antigens. Very small to negligible numbers of cells expressed markers of dendritic cells (CD11c) or granulocytes (Ly6G). Markers for T-cells and NK cells were absent from the corneal stroma, indicating that all the cells identified in the stroma were of the myeloid lineage.

Conclusions

The normal murine corneal stroma contains a significant number of CD45+ leukocytes. Most these cells express the CD11b marker, but not other dendrite, granulocyte, T-cell, or NK markers, placing them in the monocyte/macrophage lineage.

Purpose

To determine whether lacrimal and salivary gland nerves of an animal model of Sjögren's syndrome, the MRL/lpr mouse, are able to release acetylcholine. The second purpose was to determine whether activation of the lacrimal gland nerves of the MRL/lpr mouse leads to protein secretion.

Methods

Total saliva was collected for 10 minutes from the oral cavity of male and female MRL/lpr and MRL/+ mice, after intraperitoneal stimulation with pilocarpine and isoproterenol. Lacrimal and salivary gland lobules prepared from 18-week-old MRL/lpr and MRL/+ mice were incubated in the presence of depolarizing KCl (75 mM) solution. Acetylcholine release and peroxidase secretion (a protein secreted by the lacrimal gland) were measured using a spectrofluorometric assay.

Results

Female, but not male, MRL/lpr mouse salivary glands were hyper-responsive to in vivo injection of secretagogues. These mice produced significantly higher amounts of saliva than did age-matched MRL/+ mice. Lacrimal and salivary gland nerves from 18-week-old MRL/+ mice released acetylcholine in response to a depolarizing KCl solution. In contrast, nerves in glands from 18-week-old MRL/lpr mice did not increase acetylcholine release in response to the depolarizing solution. Moreover, lacrimal glands from 18-week-old MRL/+ mice were able to secrete peroxidase in response to a depolarizing KCl solution, whereas those from 18-week-old MRL/lpr could not. This was not due to a defect in the secretory process, because addition of an exogenous secretagogue elicited peroxidase secretion from 18-week-old MRL/lpr as well as MRL/+ mice lacrimal glands.

Conclusions

The results show that activation of nerves of lacrimal and salivary glands infiltrated with lymphocytes does not increase the release of neurotransmitters, which results in impaired secretion from these glands. (Invest Ophthalmol Vis Sci.

Purpose

To determine the effects of the proinflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α on neurally mediated lacrimal gland protein secretion and to determine whether the amount of IL-1β protein is upregulated in inflamed lacrimal glands of the MRL/lpr mouse, a murine model of human Sjögren syndrome.

Methods

Lacrimal gland lobules of BALB/c mice were prepared and incubated for 2 hours in the presence or absence of recombinant human (rh)IL-1α, rhIL-1β (10 ng/mL each), or rhTNFα (50 ng/mL). Peroxidase secretion in response to depolarizing KCl (75 mM) solution was measured by spectrofluorometric assay. In another set of experiments, saline, rhIL-1β(1 µg), or an antibody against IL-1 receptor type I (IL-1RI), with or without rhIL-1β, was injected (2 µL) into the lacrimal glands of anesthetized BALB/c mice. Twenty-four hours later, lacrimal gland lobules were prepared and peroxidase secretion was measured. The amount of IL-1β protein in lacrimal gland acinar cell lysates prepared from 3-, 9-, and 13-week-old BALB/c, MRL/+, and MRL/lpr mice was determined by ELISA.

Results

KCl-induced peroxidase secretion was inhibited in vitro 62%, 66%, and 53% by rhIL-1α, rhIL-1β, and rhTNFα, respectively. In vivo, rhIL-1β inhibited KCl-induced peroxidase secretion by 72%. This inhibitory effect of IL-1β was completely reversed by an antibody against IL-1RI. Compared with 3-week-old mice, the amount of IL-1β protein was upregulated 15- and 21-fold in lacrimal gland acinar cells isolated from 9- and 13-week-old MRL/lpr mice, respectively.

Conclusions

Proinflammatory cytokines inhibit neurally mediated lacrimal gland secretion. The amount of IL-1β protein is upregulated in acinar cells prepared from lacrimal glands infiltrated with lymphocytes. These results suggest that elevated levels of IL-1β, as they occur in Sjögren syndrome exocrine glands, may impair the secretory function of these tissues.

Purpose

To characterize the angiogenic and inflammatory responses of human choroidal endothelial cells (HCECs) to stimulators and inhibitors of the ubiquitin proteasome pathway (UPP).

Methods

The regulation of the UPP by the inhibitor withaferin A and its congener, withanolide D, two natural products derived from the medicinal plant Withania somnifera was assessed in the three-dimensional endothelial cell sprouting assay (3D-ECSA), by using HCEC- and human umbilical vein endothelial cell (HUVEC)– derived spheroids embedded in a collagen I matrix. Western blot analysis was used to investigate the effect of withanolides on IκB-α, polyubiquitination, and heme oxygenase (HO)-1 regulation in HCEC and HUVEC cultures.

Results

HCECs, like HUVECs, responded to fibroblast growth factor-2, vascular endothelial growth factor, and tumor necrosis factor (TNF)-α stimulation and sprouted vessel-like structures in collagen I matrix. However, HCECs were slower to generate these sprouting vessels, when compared with HUVECs. The extent of inhibition of endothelial cell sprouting in 3D matrix, the blockade of TNF-α-induced IκB-α degradation, levels of global polyubiquitinated proteins, and induced production of HO-1 in response to treatment by the withanolides in cultured endothelial cells was similarly regulated between HCECs and HUVECs.

Conclusions

HCECs share with HUVECs a similar response to UPP inhibitors, suggesting that this well-conserved pathway that regulates angioinflammatory mechanisms could be exploited for drug-targeting in the development of novel agents for CNV treatment.

Purpose

To investigate the expression and regulation of the short form of caspase-12, caspase-12S, in human retinal pigment epithelial (hRPE) cells.

Methods

hRPE cells were stimulated by pro-inflammatory agents IL-1β and TNF-α, LPS, co-culture with monocytes, immunomodulating agent cyclosporine A (CSA), and anti-inflammatory cytokine IL-10, and endoplasmic reticulum (ER) stress inducers tunicamycin and thapsigargin for 6 hr or longer. The total RNAs were isolated and subjected to semi-quantitative and quantitative real time RT-PCR analysis. Effects of tunicamycin and thapsigargin on IL-1β– and TNF-α-stimulated MCP-1 mRNA expression and protein production were further examined by RT-PCR and ELISA, respectively.

Results

RT-PCR results showed that caspase-12S is the predominant form of caspase-12 in the examined hRPE cells of this study with expression at as high levels as in many other human tissues such as pancreas, prostate, small intestine, lung, spleen, and kidney. Treatment with IL-1β and TNF-α, as well as LPS and co-culture with monocytes reduced hRPE caspase-12S mRNA expression within 6 hr. In contrast, hRPE exposure to cyclosporine A (CSA) and cytokine IL-10 for 6 hr increased caspase-12S mRNA expression. Compared to CSA and IL-10, ER stress activators tunicamycin and thapsigargin were even more potent enhancers of hRPE caspase-12S gene expression. Tunicamycin and thapsigargin also caused corresponding reductions in IL-1β- and TNF-α-induced MCP-1 mRNA expression and protein production.

Conclusion

hRPE cells express high level of caspase-12S. The regulated expression of caspase-12S suggests that the caspase recruitment domain (CARD)-only caspase-12S may be an endogenous dominant negative regulator modulating inflammatory responses in hRPE cells.