EFFICACY OF INTRAVITREAL AFLIBERCEPT INJECTIONS IN THE TREATMENT OF IDIOPATHIC RETINAL VASCULITIS, ANEURYSMS, AND NEURORETINITIS SYNDROME.

PURPOSE: To present a case of idiopathic retinal vasculitis, aneurysms, and neuroretinitis syndrome that was successfully managed with serial intravitreal aflibercept injections. METHODS: Ophthalmic imaging and visual acuity were used to monitor disease state and track treatment methods to determine the most valuable combination of treatment medication and treatment interval. RESULTS: A 28-year-old woman with idiopathic retinal vasculitis, aneurysms, and neuroretinitis syndrome status after panretinal photocoagulation of both eyes presented with bilateral cystoid macular edema. We demonstrate successful management of retinal cystoid macular edema associated with idiopathic retinal vasculitis, aneurysms, and neuroretinitis syndrome using serial intravitreal aflibercept injections. CONCLUSION: Intravitreal aflibercept has a useful role in managing the potential retinal complications associated with idiopathic retinal vasculitis, aneurysms, and neuroretinitis syndrome and provides further insights into treatment of the later stages of this rare disease.

Prostaglandin analog effects on cerebrospinal fluid reabsorption via nasal mucosa.

INTRODUCTION: Cerebrospinal fluid (CSF) outflow has been demonstrated along nasal lymphatics via olfactory nerve projections; flow may be increased by stimulating lymphatic contractility using agents such as noradrenaline and the thromboxane A2 analog U46619. Lymphatics elsewhere in the body show increased contractility upon exposure to the prostaglandin F2alpha analog isoprostane-8-epi-prostaglandin. We investigated the ability of ophthalmic prostaglandin F2alpha analogs to increase CSF outflow when applied to the nasal mucosa by inhalation. METHODS: Latanoprost (0.1, 0.5, or 1mg/ml), bimatoprost (0.3 or 3mg/ml), travoprost (0.04 or 0.4mg/ml), latanoprostene bunod (0.24 or 2.4mg/ml), tafluprost (0.25 or 2.5mg/ml), or control vehicle (10% DMSO) was administered to awake adult C57B/6 mice by nasal inhalation of 2µl droplets. Multiday dosing (daily for 3 days) of latanoprost also was evaluated. A total of 81 animals were studied including controls. General anesthesia was induced by injection, and fluorescent tracer (AlexaFluor647-labelled ovalbumin) was injected under stereotaxic guidance into the right lateral ventricle. Nasal turbinate tissue was harvested and homogenized after 1 hour for tracer detection by ELISA and fluorometric analysis. RESULTS: Inhalation of latanoprost 0.5mg/ml and 1mg/ml led to a 11.5-fold increase in tracer recovery from nasal turbinate tissues compared to controls (3312 pg/ml vs 288 pg/ml, p<0.001 for 0.5mg/ml; 3355 pg/ml vs 288 pg/ml, p<0.001 for 1mg/ml), while latanoprost 0.1 mg/ml enhanced recovery 6-fold (1713 pg/ml vs 288 pg/ml, p<0.01). Tafluprost 0.25mg/ml and bimatoprost 0.3mg/ml showed a modest (1.4x, p<0.05) effect, and the remaining agents showed no significant effect on tracer recovery. After 3 days of daily latanoprost treatment and several hours after the last dose, a persistently increased recovery of tracer was found. CONCLUSIONS: Prostaglandin F2alpha analogs delivered by nasal inhalation resulted in increased nasal recovery of a CSF fluorescent tracer, implying increased CSF outflow via the nasal lymphatics. The greatest effect, partially dose-dependent, was observed using latanoprost. Further studies are needed to determine the efficacy of these agents in reducing ICP in short and long-term applications.

Nanogel-Facilitated In-Situ Delivery of a Cataract Inhibitor.

Cataracts are a leading cause of blindness worldwide. Surgical removal of cataracts is a safe and effective procedure to restore vision. However, a large number of patients later develop vision loss due to regrowth of lens cells and subsequent degradation of the visual axis leading to visual disability. This postsurgical complication, known as posterior capsular opacification (PCO), occurs in up to 30% of cataract patients and has no clinically proven pharmacological means of prevention. Despite the availability of many compounds capable of preventing early steps in PCO development, there is currently no effective means to deliver such therapies into the eye for a suitable duration. To model a solution to this unmet medical need, we fabricated acrylic substrates as intraocular lens (IOL) mimics scaled to place into the capsular bag of the mouse lens following a mock-cataract surgery. Substrates were coated with a hydrophilic crosslinked acrylate nanogel designed to elute Sorbinil, an aldose reductase inhibitor previously shown to suppress PCO. Insertion of the Sorbinil-eluting device into the lens capsule at the time of cataract surgery resulted in substantial prevention of cellular changes associated with PCO development. This model demonstrates that a cataract inhibitor can be delivered into the postsurgical lens capsule at therapeutic levels.

The Protective Effect of Metformin Use on Early Nd:YAG Laser Capsulotomy.

Purpose: To determine the effect of metformin on early Nd:YAG laser treatment for posterior capsule opacification (PCO) and to explore a molecular mechanism to explain a possible protective effect of metformin against PCO. Methods: We conducted: 1) a retrospective cohort study of patient eyes undergoing phacoemulsification at our institution; and 2) laboratory investigation of the effect of metformin on the behavior of lens epithelial cells in the context of an animal model for PCO. Population-averaged Cox proportional hazards modeling was used to estimate risk for time to Nd:YAG. For laboratory studies, expression of markers for epithelial-to-mesenchymal transition (EMT) implicated in PCO pathogenesis was measured in tissue culture and following extracapsular lens extraction in a mouse model. Results: The rate of Nd:YAG laser capsulotomy was 13.1% among the 9798 eyes. Both metformin use and diabetes were protective factors for Nd:YAG laser capsulotomy in univariate analysis. However, in multivariable analysis with nondiabetics as the reference group, only metformin use among diabetics was significantly protective of Nd:YAG (hazard ratio: 0.68, 95% CI: 0.54-0.85, P = 0.0008), while eyes of patients with diabetes without metformin use did not significantly differ (P = 0.5026). Treatment of lens epithelial cells with metformin reduced the level of the EMT markers ⍺-SMA and pERK induced by TGF-ß2. Similarly, metformin treatment reduced ⍺-SMA expression in lens epithelial cells following extracapsular lens extraction in a mouse model. Conclusions: The protective effect of metformin against early Nd:YAG may relate to its ability to downregulate EMT in residual lens epithelial cells that otherwise trend toward myofibroblast development and PCO.

Diabetes-Independent Retinal Phenotypes in an Aldose Reductase Transgenic Mouse Model.

Aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, has been implicated in the onset and development of the ocular complications of diabetes, including cataracts and retinopathy. Despite decades of research conducted to address possible mechanisms, questions still persist in understanding if or how AR contributes to imbalances leading to diabetic eye disease. To address these questions, we created a strain of transgenic mice engineered for the overexpression of human AR (AR-Tg). In the course of monitoring these animals for age-related retinal phenotypes, we observed signs of Müller cell gliosis characterized by strong immunostaining for glial fibrillary acidic protein. In addition, we observed increased staining for Iba1, consistent with an increase in the number of retinal microglia, a marker of retinal inflammation. Compared to age-matched nontransgenic controls, AR-Tg mice showed an age-dependent loss of Brn3a-positive retinal ganglion cells and an associated decrease in PERG amplitude. Both RGC-related phenotypes were rescued in animals treated with Sorbinil in drinking water. These results support the hypothesis that increased levels of AR may be a risk factor for structural and functional changes known to accompany retinopathy in humans.

Suppression of epithelial to mesenchymal transition markers in mouse lens by a Smad7-based recombinant protein.

Cataracts, a clouding of the eye lens, are a leading cause of visual impairment and are responsible for one of the most commonly performed surgical procedures worldwide. Although generally safe and effective, cataract surgery can lead to a secondary lens abnormality due to transition of lens epithelial cells to a mesenchymal phenotype (EMT) and opacification of the posterior lens capsular bag. Occurring in up to 40% of cataract cases over time, posterior capsule opacification (PCO) introduces additional treatment costs and reduced quality of life for patients. Studies have shown that PCO pathogenesis is driven in part by TGF-ß, signaling through the action of the family of Smad coactivators to effect changes in gene transcription. In the present study, we evaluated the ability of Smad-7, a well characterized inhibitor of TGF-ß -mediated Smad signaling, to suppress the EMT response in lens epithelial cells associated with PCO pathogenesis. Treatment of lens epithelial cells with a cell-permeable form of Smad7 variant resulted in suppressed expression of EMT markers such as alpha smooth muscle actin and fibronectin. A single application of cell-permeable Smad7 variant in the capsular bag of a mouse cataract surgery model resulted in suppression of gene transcripts encoding alpha smooth muscle actin and fibronectin. These results point to Smad7 as a promising biotherapeutic agent for prevention or substantial reduction in the incidence of PCO following cataract surgery.

Aldose reductase inhibition enhances lens regeneration in mice.

After cataract surgery, epithelial cells lining the anterior lens capsule can transition to one of two divergent pathways, including fibrosis which leads to posterior capsular opacification (PCO), or lens fiber cell differentiation which leads to regeneration of lens material. We previously showed that the PCO response can be suppressed with aldose reductase (AR) inhibitors. In this present study we show that AR inhibition, both genetic and pharmacologic with Sorbinil, can augment the process of lens regeneration. Extracapsular lens extraction (ECLE) was carried out in C57BL/6 (WT), AR overexpression (AR-Tg), and AR knockout (ARKO) mice, and in some cases in mice treated with the AR inhibitor sorbinil. Whole eyes were harvested approximately 8 weeks after ECLE and evaluated by histological analysis and immunostaining for the fiber cell marker γ-crystallin. All eyes examined for lens regeneration were paraffin embedded for serial sectioning to produce three-dimensional reconstructed models of lens morphology and size. We observed that AR-null mice respond to ECLE by regenerating a lens-like structure with a circular shape and array of cell nuclei reminiscent of the lens bow region typical of the native mammalian lens. Although WT and AR-Tg eyes also produced some regenerated lens material after ECLE, their structures were consistently smaller than ARKO regenerated lenses. WT mice treated with sorbinil showed higher levels of lens regeneration after ECLE compared to WT mice, as assessed by size and three-dimensional morphology. Altogether, this study adds evidence for a critical role for AR in the response of lens epithelial cells to cataract extraction and lens regeneration.

Endothelial Cell-Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature.

Objective- Blood-CNS (central nervous system) barrier defects are implicated in retinopathies, neurodegenerative diseases, stroke, and epilepsy, yet, the pathological mechanisms downstream of barrier defects remain incompletely understood. Blood-retina barrier (BRB) formation and retinal angiogenesis require ß-catenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4 (frizzled 4), coreceptor LRP5 (low-density lipoprotein receptor-like protein 5), and the tetraspanin TSPAN12 (tetraspanin 12). Impaired NDP/FZD4 signaling causes familial exudative vitreoretinopathy, which may lead to blindness. This study seeked to define cell type-specific functions of TSPAN12 in the retina. Approach and Results- A loxP-flanked Tspan12 allele was generated and recombined in endothelial cells using a tamoxifen-inducible Cdh5-CreERT2 driver. Resulting phenotypes were documented using confocal microscopy. RNA-Seq, histopathologic analysis, and electroretinogram were performed on retinas of aged mice. We show that TSPAN12 functions in endothelial cells to promote vascular morphogenesis and BRB formation in developing mice and BRB maintenance in adult mice. Early loss of TSPAN12 in endothelial cells causes lack of intraretinal capillaries and increased VE-cadherin (CDH5 [cadherin5 aka VE-cadherin]) expression, consistent with premature vascular quiescence. Late loss of TSPAN12 strongly impairs BRB maintenance without affecting vascular morphogenesis, pericyte coverage, or perfusion. Long-term BRB defects are associated with immunoglobulin extravasation, complement deposition, cystoid edema, and impaired b-wave in electroretinograms. RNA-sequencing reveals transcriptional responses to the perturbation of the BRB, including genes involved in vascular basement membrane alterations in diabetic retinopathy. Conclusions- This study establishes mice with late endothelial cell-specific loss of Tspan12 as a model to study pathological consequences of BRB impairment in an otherwise intact vasculature.

Aldose Reductase Inhibition Prevents Development of Posterior Capsular Opacification in an In Vivo Model of Cataract Surgery.

Purpose: Cataract surgery is a procedure by which the lens fiber cell mass is removed from its capsular bag and replaced with a synthetic intraocular lens. Postoperatively, remnant lens epithelial cells can undergo an aberrant wound healing response characterized by an epithelial-to-mesenchymal transition (EMT), leading to posterior capsular opacification (PCO). Aldose reductase (AR) inhibition has been shown to decrease EMT markers in cell culture models. In this study, we aim to demonstrate that AR inhibition can attenuate induction of EMT markers in an in vivo model of cataract surgery. Methods: A modified extracapsular lens extraction (ECLE) was performed on C57BL/6 wildtype, AR overexpression (AR-Tg), and AR knockout mice. Immunofluorescent staining for the myofibroblast marker α-smooth muscle actin (α-SMA), epithelial marker E-cadherin, and lens fiber cell markers αA-crystallin and Aquaporin 0 was used to characterize postoperative PCO. Quantitative reverse transcription PCR (qRT-PCR) was employed to quantify postoperative changes in α-SMA, vimentin, fibronectin, and E-cadherin. In a separate experiment, the AR inhibitor Sorbinil was applied postoperatively and qRT-PCR was used to assess changes in EMT markers. Results: Genetic AR knockout reduced ECLE-induced upregulation of α-SMA and downregulation of E-cadherin. These immunofluorescent changes were mirrored quantitatively in changes in mRNA levels. Similarly, Sorbinil blocked characteristic postoperative EMT changes in AR-Tg mice. Interestingly, genetic AR knockout did not prevent postoperative induction of the lens fiber cell markers αA-crystallin and Aquaporin 0. Conclusions: AR inhibition prevents the postoperative changes in EMT markers characteristic of PCO yet preserves the postoperative induction of lens fiber cell markers.

The effect of amniotic membrane grafting on healing and wound strength after strabismus surgery in a rabbit model.

BACKGROUND: Amniotic membrane grafts (AMGs) are used, with mixed results, as a platform for ocular healing and to reduce pathologic scarring. This study evaluated wound tensile strength and histopathologic changes after strabismus surgery with AMGs in 20 New Zealand white rabbits. METHODS: All subjects underwent 4 mm inferior rectus hang-back recessions to both eyes. The right eyes served as controls. Ten left eyes (group 1) received processed dehydrated amniotic membrane allografts (Ambiodry2, IOP Inc, Costa Mesa, CA) and ten left eyes (group 2) received cryopreserved human amniotic membrane allografts (AmnioGraft, Bio-Tissue, Miami, FL) between the sclera and muscle insertion and between the muscle and repositioned conjunctiva. At postoperative month 1, tensile strengths of the muscle-globe and conjunctiva-globe attachments were measured, and histopathologic analysis of each eye was performed. RESULTS: In group 1 the mean tensile strength of the muscle-globe attachments was 441.4 ± 274.4 g; of the conjunctiva-globe attachments, 640.3 ± 266.4 g. In the control eyes, the comparable values were 365.8 ± 199.8 g and 595.2 ± 315.3 g, respectively (P = 0.19, P = 0.13). In group 2 the mean tensile strengths were 456 ± 297.5 g and 608.2 ± 306.7 g, compared with control values of 352.7 ± 114.8 g and 583.8 ± 347.1 g (P = 0.43, P = 0.45). CONCLUSIONS: There was no significant change in tensile strength of the muscle insertion using AMGs. In a rabbit model, AMGs do not reduce inflammation or improve scar formation 1 month after strabismus surgery.

Effect of human milk as a treatment for dry eye syndrome in a mouse model.

PURPOSE: Dry eye syndrome (DES) affects millions of people worldwide. Homeopathic remedies to treat a wide variety of ocular diseases have previously been documented in the literature, but little systematic work has been performed to validate the remedies' efficacy using accepted laboratory models of disease. The purpose of this study was to evaluate the efficacy of human milk and nopal cactus (prickly pear), two widely used homeopathic remedies, as agents to reduce pathological markers of DES. METHODS: The previously described benzalkonium chloride (BAK) dry eye mouse model was used to study the efficacy of human milk and nopal cactus (prickly pear). BAK (0.2%) was applied to the mouse ocular surface twice daily to induce dry eye pathology. Fluorescein staining was used to verify that the animals had characteristic signs of DES. After induction of DES, the animals were treated with human milk (whole and fat-reduced), nopal, nopal extract derivatives, or cyclosporine four times daily for 7 days. Punctate staining and preservation of corneal epithelial thickness, measured histologically at the end of treatment, were used as indices of therapeutic efficacy. RESULTS: Treatment with BAK reduced the mean corneal epithelial thickness from 36.77±0.64 µm in the control mice to 21.29±3.2 µm. Reduction in corneal epithelial thickness was largely prevented by administration of whole milk (33.2±2.5 µm) or fat-reduced milk (36.1±1.58 µm), outcomes that were similar to treatment with cyclosporine (38.52±2.47 µm), a standard in current dry eye therapy. In contrast, crude or filtered nopal extracts were ineffective at preventing BAK-induced loss of corneal epithelial thickness (24.76±1.78 µm and 27.99±2.75 µm, respectively), as were solvents used in the extraction of nopal materials (26.53±1.46 µm for ethyl acetate, 21.59±5.87 µm for methanol). Epithelial damage, as reflected in the punctate scores, decreased over 4 days of treatment with whole and fat-reduced milk but continued to increase in eyes treated with nopal-derived materials. CONCLUSIONS: Whole and fat-reduced human milk showed promising effects in the prevention of BAK-induced loss of corneal epithelial thickness and epithelial damage in this mouse model. Further studies are required to determine whether human milk may be safely used to treat dry eye in patients.

Impact of Subunit Composition on the Uptake of α-Crystallin by Lens and Retina.

Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. α-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with α-crystallin may help to delay disease onset. The purpose of this study was to determine if αB-crystallin subunits containing a cell penetration peptide (gC-tagged αB-crystallin) facilitate the uptake of wild type αA-crystallin (WT-αA) in lens and retina. Recombinant human αB-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-αB). Recombinant gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37°C for 4 hours to allow for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ cultures. Similarly, crystallin mixtures were injected into the vitreous of rat eyes. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity assays were performed on α-crystallins ratios showing optimal uptake using chemically-induced or heat induced substrate aggregation assays. As determined by flow cytometry, a ratio of 1:5 for gC-αB to WT-αA was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-αB to WT-αA (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-αB to WT-αA (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of α-crystallin were found in lens and retina following intravitreal injection of homo- and hetero-oligomers in rats.

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.