High incidence of sebaceous gland inflammation in aldose reductase-deficient mice.

Aldose reductase is a member of the 1B1 subfamily of aldo-keto reductase gene superfamily. The action of aldose reductase (AR) has been implicated in the pathogenesis of a variety of disease states, most notably complications of diabetes mellitus including neuropathy, retinopathy, nephropathy, and cataracts. To explore for mechanistic roles for AR in disease pathogenesis, we established mutant strains produced using Crispr-Cas9 to inactivate the AKR1B3 gene in C57BL6 mice. Phenotyping AR-knock out (ARKO) strains confirmed previous reports of reduced accumulation of tissue sorbitol levels. Lens epithelial cells in ARKO mice showed markedly reduced epithelial-to-mesenchymal transition following lens extraction in a surgical model of cataract and posterior capsule opacification. A previously unreported phenotype of preputial sebaceous gland swelling was observed frequently in male ARKO mice homozygous for the mutant AKR1B3 allele. This condition, which was shown to be accompanied by infiltration of proinflammatory CD3+ lymphocytes, was not observed in WT mice or mice heterozygous for the mutant allele. Despite this condition, reproductive fitness of the ARKO strain was indistinguishable from WT mice housed under identical conditions. These studies establish the utility of a new strain of AKR1B3-null mice created to support mechanistic studies of cataract and diabetic eye disease.

Mitigation of lens opacification by a functional food in a diabetic rodent model.

The current study was designed to test a functional food (FF) mixture containing aldose reductase inhibitors and antiglycation bioactive compounds for suppressing the onset and progression of cataracts in a diabetic rat model. Two-month-old Sprague Dawley rats were grouped as control (C), diabetes untreated (D), and diabetic rats treated with FF at two doses (FF1 = 1.35 g and FF2 = 6.25 g/100g of diet). Diabetes was induced by a single injection of streptozotocin. The FF is a mixture of amla, turmeric, black pepper, cinnamon, ginger, and fenugreek added to the rodent diet. The status of cataracts was monitored weekly by a slit lamp examination for 20 weeks, after which animals were sacrificed to collect eye lenses. Feeding FF1 and FF2 to diabetic rats yielded a significant anti-hyperglycaemic effect and marginally prevented body weight loss. FF delayed cataract progression, and FF2 showed better efficacy than FF1. FF prevented the loss of lens crystallins and their insolubilization in diabetic rats. The antioxidant potential of FF was evident with the lowered protein carbonyls, lipid peroxidation, and prevention of altered antioxidant enzyme activities induced by diabetes. These studies demonstrate the efficacy of plant-derived dietary supplements against the onset and progression of cataracts in a well-established rat model of diabetic eye disease.

Dexamethasone targets actin cytoskeleton signaling and inflammatory mediators to reverse sulfur mustard-induced toxicity in rabbit corneas.

PURPOSE: Sulfur mustard (SM), a bi-functional alkylating agent, was used during World War I and the Iran-Iraq war. SM toxicity is ten times higher in eyes than in other tissues. Cornea is exceptionally susceptible to SM-injuries due to its anterior positioning and mucous-aqueous interphase. Ocular SM exposure induces blepharitis, photosensitivity, dry eye, epithelial defects, limbal ischemia and stem cell deficiency, and mustard gas keratopathy leading to temporary or permanent vision impairments. We demonstrated that dexamethasone (Dex) is a potent therapeutic intervention against SM-induced corneal injuries; however, its mechanism of action is not well known. Investigations employing proteomic profiling (LC-MS/MS) to understand molecular mechanisms behind SM-induced corneal injury and Dex efficacy were performed in the rabbit cornea exposed to SM and then received Dex treatment. PEAKS studio was used to extract, search, and summarize peptide identity. Ingenuity Pathway Analysis was used for pathway identification. Validation was performed using immunofluorescence. One-Way ANOVA (FDR < 0.05; p < 0.005) and Student's t-test (p < 0.05) were utilized for analyzing proteomics and IF data, respectively. Proteomic analysis revealed that SM-exposure upregulated tissue repair pathways, particularly actin cytoskeleton signaling and inflammation. Prominently dysregulated proteins included lipocalin2, coronin1A, actin-related protein2, actin-related protein2/3 complex subunit2, actin-related protein2/3 complex subunit4, cell division cycle42, ezrin, bradykinin/kininogen1, moesin, and profilin. Upregulated actin cytoskeleton signaling increases F-actin formation, dysregulating cell shape and motility. Dex reversed SM-induced increases in the aforementioned proteins levels to near control expression profiles. Dex aids corneal wound healing and improves corneal integrity via actin cytoskeletal signaling and anti-inflammatory effects following SM-induced injuries.

Establishing a Dexamethasone Treatment Regimen To Alleviate Sulfur Mustard-Induced Corneal Injuries in a Rabbit Model.

Sulfur mustard (SM) is an ominous chemical warfare agent. Eyes are extremely susceptible to SM toxicity; injuries include inflammation, fibrosis, neovascularization (NV), and vision impairment/blindness, depending on the exposure dosage. Effective countermeasures against ocular SM toxicity remain elusive and are warranted during conflicts/terrorist activities and accidental exposures. We previously determined that dexamethasone (DEX) effectively counters corneal nitrogen mustard toxicity and that the 2-hour postexposure therapeutic window is most beneficial. Here, the efficacy of two DEX dosing frequencies [i.e., every 8 or 12 hours (initiated, as previously established, 2 hours after exposure)] until 28 days after SM exposure was assessed. Furthermore, sustained effects of DEX treatments were observed up to day 56 after SM exposure. Corneal clinical assessments (thickness, opacity, ulceration, and NV) were performed at the day 14, 28, 42, and 56 post-SM exposure time points. Histopathological assessments of corneal injuries (corneal thickness, epithelial degradation, epithelial-stromal separation, inflammatory cell, and blood vessel counts) using H&E staining and molecular assessments (COX-2, MMP-9, VEGF, and SPARC expressions) were performed at days 28, 42, and 56 after SM exposure. Statistical significance was assessed using two-way ANOVA, with Holm-Sidak post hoc pairwise multiple comparisons; significance was established if P < 0.05 (data represented as the mean ± S.E.M.). DEX administration every 8 hours was more potent than every 12 hours in reversing ocular SM injury, with the most pronounced effects observed at days 28 and 42 after SM exposure. These comprehensive results are novel and provide a comprehensive DEX treatment regimen (therapeutic-window and dosing-frequency) for counteracting SM-induced corneal injuries. SIGNIFICANCE STATEMENT: The study aims to establish a dexamethasone (DEX) treatment regimen by comparing the efficacy of DEX administration at 12 versus 8 hours initiated 2 hours after exposure. DEX administration every 8 hours was more effective in reversing sulfur mustard (SM)-induced corneal injuries. SM injury reversal during DEX administration (initial 28 days after exposure) and sustained [further 28 days after cessation of DEX administration (i.e., up to 56 days after exposure)] effects were assessed using clinical, pathophysiological, and molecular biomarkers.

Nitrogen Mustard-Induced Ex Vivo Human Cornea Injury Model and Therapeutic Intervention by Dexamethasone.

Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [∼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7).

Metabolomics for identifying pathways involved in vesicating agent lewisite-induced corneal injury.

Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.

Ocular injury progression and cornea histopathology from chloropicrin vapor exposure: Relevant clinical biomarkers in mice.

Ocular tissue is highly sensitive to chemical exposures. Chloropicrin (CP), a choking agent employed during World War I and currently a popular pesticide and fumigating agent, is a potential chemical threat agent. Accidental, occupational, or intentional exposure to CP results in severe ocular injury, especially to the cornea; however, studies on ocular injury progression and underlying mechanisms in a relevant in vivo animal model are lacking. This has impaired the development of effective therapies to treat the acute and long-term ocular toxicity of CP. To study the in vivo clinical and biological effects of CP ocular exposure, we tested different CP exposure doses and durations in mice. These exposures will aid in the study of acute ocular injury and its progression as well as identify a moderate dose to develop a relevant rodent ocular injury model with CP. The left eyes of male BALB/c mice were exposed to CP (20% CP for 0.5 or 1 min or 10% CP for 1 min) using a vapor cap, with the right eyes serving as controls. Injury progression was evaluated for 25 days post-exposure. CP-exposure caused a significant corneal ulceration and eyelid swelling which resolved by day 14 post exposure. In addition, CP-exposure caused significant corneal opacity and neovascularization. Development of hydrops (severe corneal edema with corneal bullae) and hyphema (blood accumulation in the anterior chamber) was observed as advanced CP effects. Mice were euthanized at day 25 post-CP-exposure, and the eyes were harvested to further study the corneal injury. Histopathological analyses showed a significant CP-induced decrease in corneal epithelial thickness and increased stromal thickness with more pronounced damage, including stromal fibrosis, edema, neovascularization, trapped epithelial cells, anterior and posterior synechiae, and infiltration of inflammatory cells. Loss of the corneal endothelial cells and Descemet's membrane could be associated with the CP-induced corneal edema and hydrops which could lead to long term term pathological conditions. Although exposure to 20% CP for 1 min caused more eyelid swelling, ulceration, and hyphema, similar effects were observed with all CP exposures. These novel findings following CP ocular exposure in a mouse model outline the corneal histopathologic changes that associate with the continuing ocular clinical effects. The data are useful in designing further studies to identify and correlate the clinical and biological markers of CP ocular injury progression with acute and long-term toxic effects on cornea and other ocular tissues. We take a crucial step towards CP ocular injury model development and in pathophysiological studies to identify molecular targets for therapeutic interventions.

Nano-assemblies enhance chaperone activity, stability, and delivery of alpha B-crystallin-D3 (αB-D3).

Crystallins, small heat shock chaperone proteins that prevent protein aggregation, are of potential value in treating protein aggregation disorders. However, their therapeutic use is limited by their low potency and poor intracellular delivery. One approach to facilitate the development of crystallins is to improve their activity, stability, and delivery. In this study, zinc addition to αB-crystallin-D3 (αB-D3) formed supramolecular nano- and micro- assemblies, induced dose-dependent changes in structure (beta-sheet to alpha-helix) and increased surface hydrophobicity and chemical stability. Further, crystallin assemblies exhibited a size-dependent chaperone activity, with the nano-assemblies being superior to micro-assemblies and 4.3-fold more effective than the native protein in preventing ß-mercaptoethanol induced aggregation of insulin. Insulin rescued by crystallin assemblies retained the activity as evidenced by glucose uptake in 3T3-L1 cells. The most active nano-assemblies enhanced protein stability, in the presence of urea, by 1.6-fold, whereas intracellular delivery was enhanced by 3.0-fold. The αB-D3 crystallin nano-assemblies exhibit uniquely enhanced stability, activity, and delivery compared to the native protein.

Effect of dexamethasone treatment at variable therapeutic windows in reversing nitrogen mustard-induced corneal injuries in rabbit ocular in vivo model.

Nitrogen mustard (NM) is an analogue of the potent vesicating agent sulfur mustard, with well-established ocular injury models in rabbit eyes to study vesicant-induced ocular toxicity. The effects of NM-exposure to eyes may include irritation, redness, inflammation, fibrosis, epithelial degradation, blurred vision, partial/complete blindness, which may be temporary or permanent, depending on the route, duration, and dosage of exposure. Effective countermeasures against vesicant exposure are presently not available and are warranted in case of any terrorist activity or accidental leakage from stockpiles. Herein, our focus was to evaluate whether dexamethasone (DEX), an FDA approved potent corticosteroid with documented anti-inflammatory activities, could be an effective treatment modality. Accordingly, utilizing NM-induced corneal injuries in rabbit ocular in vivo model, we examined and compared the efficacy of DEX treatments when administration was started at early (2 h), intermediate (4 h), and late (6 h) therapeutic windows of intervention after NM-exposure and administered every 8 h thereafter. The effects of NM-exposure and DEX treatments were evaluated on clinical (corneal opacity, ulceration, and neovascularization), biological (epithelial thickness, epithelial-stromal separation, blood vessels density, and inflammatory cell and keratocyte counts) and molecular (COX-2 and VEGF expression) parameters, at day 1, 3, 7 and 14. Results indicated that DEX treatment markedly and effectively reversed the NM-induced injury markers in rabbit corneas. Early administration of DEX at 2 h was found to be most effective in reversing NM-induced corneal injuries, followed by DEX 4 h and DEX 6 h administration initiation, indicating that DEX has best efficacy at the early therapeutic window in our study model.

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.

Cataract inhibitors: Present needs and future challenges.

Cataracts result from opacification of the ocular lens and represent the leading cause of blindness worldwide. After surgical removal of the diseased lens material and implantation of an artificial intraocular lens, up to 50% of cataract patients develop a secondary lens defect called posterior capsular opacification (PCO). While vision can be restored in PCO patients by a laser-mediated capsulotomy, novel therapies involving inhibition of aldose reductase are now being developed to prevent PCO development and complications of laser capsulotomy. A question we wished to address was whether cataract surgeons believe there is an unmet need for a preventative PCO therapy, whether they would prescribe such a therapy were it available, and to assess their perceptions regarding the benefits of and obstacles to adopting novel PCO therapies in the place of laser capsulotomy. We gathered perspectives from adult, pediatric, and veterinary cataract surgeons using an online questionnaire. From 161 surgeon responses, we found that the majority of adult, pediatric, and veterinary cataract surgeons (78% n = 35, 88% n = 37, and 96% n = 71 respectively) believed there is an unmet need for preventative PCO therapy, with more than 95% expressing interest in incorporating such therapy into surgical protocols. Perceived benefits included optimizing visual outcomes, avoiding the need for additional procedures, eliminating complications related to neodymium:yttrium-aluminum-garnet laser, preserving the posterior capsule particularly in patients receiving multifocal intraocular lens implants, providing a viable solution for PCO in animals, and using it in developing countries that lack access to neodymium:yttrium-aluminum-garnet lasers. Perceived obstacles included potential lack of reimbursement by insurance companies, and the need for strong efficacy and safety profiles. Among adult surgeons, 70% (n = 31) indicated that preventative PCO therapy could add value to premium intraocular lens packages. Our studies revealed that cataract surgeons overwhelmingly support the development of preventative PCO therapy, and that clinical trials will play a critical role to test the safety and efficacy of specific therapeutic agents.

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.

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.

Acute corneal injury in rabbits following nitrogen mustard ocular exposure.

Sulfur mustard (SM), a potent vesicating chemical warfare agent, and its analog nitrogen mustard (NM), are both strong bi-functional alkylating agents. Eyes, skin, and the respiratory system are the main targets of SM and NM exposure; however, ocular tissue is most sensitive, resulting in severe ocular injury. The mechanism of ocular injury from vesicating agents' exposure is not completely understood. To understand the injury mechanism from exposure to vesicating agents, NM has been previously employed in our toxicity studies on primary human corneal epithelial cells and ex vivo rabbit cornea organ culture model. In the current study, corneal toxicity from NM ocular exposure (1%) was analyzed for up to 28 days post-exposure in New Zealand White male rabbits to develop an acute corneal injury model. NM exposure led to conjunctival and eyelid swelling within a few hours after exposure, in addition to significant corneal opacity and ulceration. An increase in total corneal thickness and epithelial degradation was observed starting at day 3 post-NM exposure, which was maximal at day 14 post-exposure and did not resolve until 28 days post-exposure. There was an NM-induced increase in the number of blood vessels and inflammatory cells, and a decrease in keratocytes in the corneal stroma. NM exposure resulted in increased expression levels of cyclooxygenase-2, Interleukin-8, vascular endothelial growth factor and Matrix Metalloproteinase 9 indicating their involvement in NM-induced corneal injury. These clinical, biological, and molecular markers could be useful for the evaluation of acute corneal injury and to screen for therapies against NM- and SM-induced ocular injury.

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.

Role of aldose reductase in diabetes-induced retinal microglia activation.

Diabetes-induced hyperglycemia plays a key pathogenic role in degenerative retinal diseases. In diabetic hyperglycemia, aldose reductase (AR) is elevated and linked to the pathogenesis of diabetic retinopathy (DR) and cataract. Retinal microglia (RMG), the resident immune cells in the retina, are thought to contribute to the proinflammatory phenotype in the diabetic eye. However, we have a limited understanding of the potential role of AR expressed in RMG as a mediator of inflammation in the diabetic retina. Glycated proteins accumulate in diabetes, including Amadori-glycated albumin (AGA) which has been shown to induce a proinflammatory phenotype in various tissues. In this study, we investigated the ability of AGA to stimulate inflammatory changes to RMG and macrophages, and whether AR plays a role in this process. In macrophages, treatment with an AR inhibitor (Sorbinil) or genetic knockdown of AR lowered AGA-induced TNF-α secretion (56% and 40%, respectively) as well as cell migration. In a mouse RMG model, AR inhibition attenuated AGA-induced TNF-α secretion and cell migration (67% and 40%, respectively). To further mimic the diabetic milieu in retina, we cultured RMG under conditions of hypoxia and observed the induction of TNF-α and VEGF protein expression. Downregulation of AR in either a pharmacological or genetic manner prevented hypoxia-induced TNF-α and VEGF expression. In our animal study, increased numbers of RMG observed in streptozotocin (STZ)-induced diabetic retina was substantially lower when diabetes was induced in AR knockout mice. Thus, in vitro and in vivo studies demonstrated that AR is involved in diabetes-induced RMG activation, providing a rationale for targeting AR as a therapeutic strategy for DR.

Aldo-Keto Reductases: Multifunctional Proteins as Therapeutic Targets in Diabetes and Inflammatory Disease.

Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase that has been shown to be involved in the pathogenesis of several blinding diseases such as uveitis, diabetic retinopathy (DR) and cataract. However, possible mechanisms linking the action of AR to these diseases are not well understood. As DR and cataract are among the leading causes of blindness in the world, there is an urgent need to explore therapeutic strategies to prevent or delay their onset. Studies with AR inhibitors and gene-targeted mice have demonstrated that the action of AR is also linked to cancer onset and progression. In this review we examine possible mechanisms that relate AR to molecular signaling cascades and thus explain why AR inhibition is an effective strategy against colon cancer as well as diseases of the eye such as uveitis, cataract, and retinopathy.

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.