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Visual disorders are common even after mild traumatic brain injury (mTBI) or blast exposure. The cost of blast-induced vision loss in civilians, military personnel, and veterans is significant. The visual consequences of blasts associated with TBI are elusive. Active military personnel and veterans report various ocular pathologies including corneal disorders post-combat blasts. The wars and conflicts in Afghanistan, Iraq, Syria, and Ukraine have significantly increased the number of corneal and other ocular disorders among military personnel and veterans. Binocular vision, visual fields, and other visual functions could be impaired following blast-mediated TBI. Blast-associated injuries can cause visual disturbances, binocular system problems, and visual loss. About 25% of veterans exposed to blasts report corneal injury. Blast exposure induces corneal edema, corneal opacity, increased corneal thickness, damage of corneal epithelium, corneal abrasions, and stromal and endothelial abnormality including altered endothelial density, immune cell infiltration, corneal neovascularization, Descemet membrane rupture, and increased pain mediators in animal models and the blast-exposed military personnel including veterans. Immune response exacerbates blast-induced ocular injury. TBI is associated with dry eyes and pain in veterans. Subjects exposed to blasts that cause TBI should undergo immediate clinical visual and ocular examinations. Delayed visual care may lead to progressive vision loss, lengthening/impairing rehabilitation and ultimately may lead to permanent vision problems and blindness. Open-field blast exposure could induce corneal injuries and immune responses in the cornea. Further studies are warranted to understand corneal pathology after blast exposure. A review of current advancements in blast-induced corneal injury will help elucidate novel targets for potential therapeutic options. This review discusses the impact of blast exposure-associated corneal disorders. • Active military personnel, veterans and civilians are exposed to various blasts. • Blasts induce traumatic brain and eye injury leading to visual dysfunctions. • Blasts affect corneal function leading to acute and chronic corneal damage and vision loss. • Humans exposed to blast or TBI show corneal injury causing vision impairment. [ABSTRACT FROM AUTHOR]

Abstract Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48 h and then treated with decorin (250 nM) in the presence or absence of EGF (100 ng/ml) for various time points (10–60 min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10 min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-β-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing. Graphical abstract Image 1 Highlights • Epidermal growth factor receptor (EGFR) regulates stromal repair by controlling migration and proliferation of keratocytes and fibroblasts. • Decorin acts as a biological ligand for EGFRand significantly downregulates migration of human corneal fibroblasts during wound healing. • Decorin-induced EGFR internalization occurs via caveolar-mediated endocytosis. [ABSTRACT FROM AUTHOR]

Inhibitor of differentiation (Id) proteins are DNA-binding transcription factors involved in cellular proliferation, migration, inflammation, angiogenesis and fibrosis. However, their expression and role in the cornea is unknown. The present study was undertaken to characterize the expression of Id proteins and their interactions with the pro-fibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and anti-fibrotic cytokine, bone morphogenic protein 7 (BMP7) in human cornea. Human donor corneas procured from Eye Bank were used. Id proteins were localized in human corneal sections using immunofluorescence. Primary cultures of human corneal fibroblasts (HCF) were established and treated with either TGFβ1 (5 ng/ml) or BMP7 (10 ng/ml) for 24 h in serum free medium. Expression of Id's in response to TGFβ1, BMP7 and TGFβ1 + BMP7 was analyzed by quantitative real time PCR (qRT-PCR) and western blot analysis. Id1 and Id2 proteins were ubiquitously expressed in the epithelial cells and stromal keratocytes in human cornea. The Id1 was localized to the basal epithelial cells as seen by immunohistochemistry. HCF expressed all known mammalian Id genes (Id1–Id4). In addition, Id1 and Id2 are selectively expressed in HCF. Treatment of human recombinant TGFβ1 (5 ng/ml) to serum-starved HCF showed a significant increase in Id genes (Id1, Id2 and Id4) at 2 h time point compared to BMP7 treatment, which showed time dependent increase in the expression of Id1–Id3 at 24–48 h. Combined treatment with TGFβ1 + BMP7 to HCF showed a significant increase in Id1 transcript and an increasing trend in Id3 and Id4 expression. The results of this study suggest that Id family of genes (Id1–Id4) are localized in the human cornea and expressed in the corneal fibroblasts. Also, Id's were differentially regulated with TGFβ1 and/or BMP7 in a time dependent manner and might serve as a therapeutic target in corneal fibrosis. [ABSTRACT FROM AUTHOR]

The cornea is a specialized, transparent, avascular, immune-privileged, and heavily innervated tissue that affords 2/3rd of refraction to the eye. Ocular injuries, infections, and genetic factors affect corneal function and cause vision impairment. Presently, a variety of laser/non-laser surgeries, immunosuppressants, and/or corneal transplants are predominantly used to revive sight in human patients. The development of novel, precision-guided, and tissue-targeted non-surgical therapies promoting corneal repair and regeneration based on mechanistic understanding is of paramount importance to reduce the impact of global blindness. Research over the past decade revealed that modulation of pathological signaling pathways and factors by a variety of therapeutic delivery methods effectively treats corneal disorders including corneal scar/haze, inflammation, and angiogenesis in various pre-clinical animal models and are primed for human translation. This review discusses recent advances in the areas of corneal repair, restoration, and regeneration. Herein, we provide an overview of evolving approaches and therapeutic modalities that have shown great promise in reviving corneal transparency and function through the use of small drug molecules, gene therapy, nanomedicine, stem cells, trophic factors, exosomes, stromal equivalents, bioengineered stromal scaffolds, tissue adhesives, and 3D bioprinting. [ABSTRACT FROM AUTHOR]

Abstract: Successful restoration of vision in human patients with gene therapy affirmed its promise to cure ocular diseases and disorders. The efficacy of gene therapy is contingent upon vector and mode of therapeutic DNA introduction into targeted cells/tissues. The cornea is an ideal tissue for gene therapy due to its ease of access and relative immune-privilege. Considerable progress has been made in the field of corneal gene therapy in last 5 years. Several new gene transfer vectors, techniques and approaches have evolved. Although corneal gene therapy is still in its early stages of development, the potential of gene-based interventions to treat corneal abnormalities has begun to surface. Identification of next generation viral and nanoparticle vectors, characterization of delivered gene levels, localization, and duration in the cornea, and significant success in controlling corneal disorders, particularly fibrosis and angiogenesis, in experimental animal disease models, with no major side effects have propelled gene therapy a step closer toward establishing gene-based therapies for corneal blindness. Recently, researchers have assessed the delivery of therapeutic genes for corneal diseases and disorders due to trauma, infections, chemical, mechanical, and surgical injury, and/or abnormal wound healing. This review provides an update on the developments in gene therapy for corneal diseases and discusses the barriers that hinder its utilization for delivering genes in the cornea. [Copyright &y& Elsevier]

Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 μl; 5x1012 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57-65, p<0.5), and CD31 immunoblotting (62-67%, p<0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5-mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients. [ABSTRACT FROM AUTHOR]

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slitlamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients. [ABSTRACT FROM AUTHOR]

Abstract: Decorin, a small leucine-rich proteoglycan, is a natural inhibitor of transforming growth factor beta (TGFβ). Myofibroblast and haze formation in the cornea have been attributed to TGFβ hyperactivity released from corneal epithelium following injury to eye. This study tested the hypothesis that decorin-gene transfer inhibits TGFβ-driven myofibroblast and haze formation in the cornea. Human corneal fibroblast (HSF) cultures generated from donor human corneas were used. Decorin cDNA was cloned into mammalian expression vector. Restriction enzyme analysis and DNA sequencing confirmed the nucleotide sequence of generated vector construct. The decorin gene cloned into mammalian expression vector was introduced into HSF with lipofectamine transfection kit. Expression of decorin in selected clones was characterized with RT-PCR, immunocytochemistry and western blotting. Phage contrast microscopy and trypan blue exclusion assay evaluated the effects of decorin-gene transfer on HSF phenotype and viability, respectively. Real-time PCR, western blot and immunocytochemistry were used to analyze inhibitory effects of decorin-gene transfer on TGFβ-induced myofibroblast formation by measuring differential expression of alpha smooth muscle actin (SMA), a myofibroblast marker, mRNA and protein expression. Analysis of variance (ANOVA) and the Bonferroni–Dunn adjustment for repeated measures were used for statistical analysis. Our data indicate that decorin-gene transfer into HSF do not alter cellular phenotype or viability. Decorin over-expressing HSF clones grown in the presence of TGFβ1 under serum-free conditions showed a statistically significant 80–83% decrease in SMA expression (p value < 0.01) compared to naked-vector transfected clones or un-transfected HSF controls. Decorin-transfected, naked-vector transfected and un-transfected HSF grown in the absence of TGFβ1 showed no or extremely low expression of SMA. Furthermore, decorin over-expression did not affect HSF phenotype and decreased TGFβ-induced RNA levels of profibrogenic genes such as fibronectin, collagen type I, III, and IV that play important role in stromal matrix modulation and corneal wound healing. The results of study suggest that decorin-gene transfer effectively prevents TGFβ-driven transformation of keratocyte and corneal fibroblast to myofibroblasts. We postulate that decorin-gene therapy can be used to treat corneal haze in vivo. [Copyright &y& Elsevier]

Abstract: Refractive surgery is a popular method used to reduce or eliminate dependence on glasses and contact lenses. Corneal haze is one of the common complications observed after photorefractive keratectmomy (PRK). The objective of this study was to develop an in vivo mouse model that consistently produces moderate to severe corneal haze in the anterior stroma of the mouse cornea after excimer laser treatment to study myofibroblast biology and corneal wound healing in a genetically defined model. Regular- or irregular-phototherapeutic keratectomy (PTK) was performed on black C57BL/6 mice with the Summit Apex excimer laser (Alcon, Ft. Worth, TX). Different numbers of laser pulses (45; ablation depth ∼10μm) were fired on the central cornea, after scraping the epithelium prior to excimer laser ablation. Irregularity was generated by positioning a fine mesh screen in the path of laser after firing 50% of the pulses. Eyes were collected 1, 2, 3 or 4weeks after the procedure. Haze formation was gauged with slit lamp biomicroscopy. Immunocytochemistry was used to determine number of myofibroblasts in the mouse cornea using antibodies specific for the myofibroblast marker alpha-smooth muscle actin (SMA). The numbers of SMA-positive cells/400× microscopic were determined by counting within the stroma. Statistical analysis was performed using analysis of variance (AVOVA) with the Bonferonni–Dunn adjustment for repeated measures. Regular-PTK with epithelial scrape (group 3) and irregular-PTK with epithelial scrape (group 4) in the mouse eyes were performed to produce corneal haze. Eyes collected 4weeks after regular- or irregular-PTK after epithelial scrape showed 22±6.6 (group 3) or 34±7.9 (group 4) SMA-positive cells in the anterior cornea. The difference in the SMA-positive cells detected among the groups was statistically significant (p <0.01). Less than 4 SMA-positive cells were detected in the tissue sections of the mouse eyes collected after 1, 2 or 3weeks of regular (group 3) or irregular PTK (group 4) or controls (groups 1 and 2). The optimized PTK excimer laser conditions developed in this study produces haze selectively in anterior stroma of the mouse cornea immediately beneath the epithelial basement membrane. Irregular PTK performed after epithelial scrape by applying 45 laser pulses was found to be the most effective method to generate myofibroblasts. This PTK technique for inducing haze in mouse cornea in vivo provides a useful model for studying wound healing and myofibroblast biology in transgenic mice. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups: Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin α4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2±0.2 cells/400× field) and group V (1.8±0.4), with significantly more in groups II (3.5±1.8), III (6.8±1.6), VII (7.9±3.8), IV (12.4±4.2) and VI (14.6±5.1). The screened groups were significantly different from each other (p<0.05), with myofibroblast generation increasing with higher surface irregularity in the −4.5 diopter PRK groups. The −9.0 diopter PRK group VI had significantly more myofibroblast generation than the −9.0 diopter PRK with PTK-smoothing group VII (p<0.01). Areas of basement membrane disruption were demonstrated by staining corneas for integrin α4 and were prominent in corneas with grade I or higher haze. SMA-positive myofibroblasts tended to be present sub-adjacent to basement membrane defects. Late apoptosis was detected at 1 month after surgery within clusters of myofibroblasts in the sub-epithelial stroma. In conclusion, these results demonstrated a relationship between the level of corneal haze formation after PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane regeneration and increased epithelium-derived TGFβ signalling to the stroma that increases myofibroblast generation. Late apoptosis appears to have a role in the disappearance of myofibroblasts and haze over time. [Copyright &y& Elsevier]

Abstract: Technological advances in the field of gene therapy has prompted more than three hundred phase I and phase II gene-based clinical trials for the treatment of cancer, AIDS, macular degeneration, cardiovascular, and other monogenic diseases. Besides treating diseases, gene transfer technology has been utilized for the development of preventive and therapeutic vaccines for malaria, tuberculosis, hepatitis A, B and C viruses, AIDS, and influenza. The potential therapeutic applications of gene transfer technology are enormous. The cornea is an excellent candidate for gene therapy because of its accessibility and immune-privileged nature. In the last two decades, various viral vectors, such as adeno, adeno-associated, retro, lenti, and herpes simplex, as well as non-viral methods, were examined for introducing DNA into corneal cells in vitro, in vivo and ex vivo. Most of these studies used fluorescent or non-fluorescent marker genes to track the level and duration of transgene expression in corneal cells. However, limited studies were directed to evaluate prospects of gene-based interventions for corneal diseases or disorders such as allograft rejection, laser-induced post-operative haze, herpes simplex keratitis, and wound healing in animal models. We will review the successes and obstacles impeding gene therapy approaches used for delivering genes into the cornea. [Copyright &y& Elsevier]

Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/−) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/− of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/− of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air–liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures. [Copyright &y& Elsevier]

Development of gene transfer methods that can precisely deliver therapeutic genes to the localized or targeted tissue(s) would be highly beneficial in developing new gene therapy approaches and may also extend animal models for studying in vivo gene function and regulation at molecular levels in the selected tissues. We investigated lipid- and AAV-mediated gene transfer in rabbit cornea using a lamellar flap-technique. The goals of this study were to (1) analyze methods for in situ gene transfer into keratocytes, (2) identify efficient and suitable vectors for gene transfer into keratocytes, and (3) characterize times of first detectable expression, localization and duration of transgene expression in keratocytes with different vectors. A lamellar flap was produced in the rabbit cornea with a microkeratome. Recombinant adeno-associated viral vector (rAAV) expressing either β-galactosidase (rAAV-β-gal) or chloramphenicol acetyltransferase (rAAV–CAT) reporter genes, or plasmid-cationic lipid complexes expressing CAT (pMP6-CAT) or β-galactosidase (pTR-β-gal) were applied beneath the lamellar flap for two minutes. The flap was repositioned and eyelids sutured overnight. Corneas were removed at 4 hr, 12 hr, 36 hr, 3 days, 7 days, or 10 days after application and either fixed in 2% formaldehyde, cryosectioned and stained for β-galactosidase activity or homogenized and measured for CAT levels by ELISA. Corneas infected with rAAV-β-gal vector showed positive β-gal staining in the center and periphery of the flap interface in whole corneas and corneal beds at 3, 7, and 10 days, but not at earlier time points. Corneas treated with pTR-β-gal plasmid vector showed positive β-gal expression at the interface at 4, 12 and 36 hr, but not at 3 or 7 days. The posterior surface of the lamellar interface where the vector was applied showed more expression than the overlying anterior surface with both plasmid and viral vectors. The level of gene expression was less with plasmid vector than viral vector monitored using β-gal staining. CAT-ELISA confirmed expression of the CAT reporter gene with either the plasmid or rAAV vector. These results demonstrate that foreign genes can be introduced into keratocytes with plasmid or viral vectors using a lamellar flap to gain access to the stroma. The expression profile of the reporter genes depended on the vector. Transfection of keratocytes with plasmid vectors produced rapid expression of the reporter genes, but for a short duration. Reporter gene expression following transduction by rAAV vector was delayed several days, but was at higher levels and for a longer duration. This is the first report to demonstrate selective gene transfer into keratocytes and would be highly useful in studying function and regulation of genes in vivo and may eventually furnish a tool for the treatment of corneal dystrophies. [Copyright &y& Elsevier]

Background Methods Results Conclusion Corneal fibrosis is a leading cause of blindness in mammalian species and may result in compromised performance in sports and daily functions. This study evaluated the safety and anti‐fibrotic effects of the FDA‐approved drugs, angiotensin‐converting enzyme inhibitor (ACE‐I) lisinopril and rho‐kinase inhibitor (ROCK‐I) fasudil, alone and in combination, on the canine cornea using an established in vitro model.To test the safety and efficacy of lisinopril and fasudil, primary canine corneal fibroblasts (CCFs) generated from donor corneas of healthy dogs (n = 20) were used. A series of dose‐dependent and time‐dependent assays with lisinopril (1–50 μM) and fasudil (1–10 nM) were performed. qRT‐PCR, immunofluorescence (IF) staining, cell viability assay, cell proliferation assay, LIVE/DEAD viability/cytotoxicity assay, TUNEL assay, and total cell count were performed.A 25‐μM lisinopril and 3‐nM fasudil dose were safe, nontoxic, and optimal for therapeutic evaluations in vitro. Treatments of lisinopril or fasudil, alone or in‐combination, to CCFs grown in the presence of TGF‐β1 (5 ng/mL) showed inhibition of myofibroblast formation based on phase‐contrast microscopy. The qRT‐PCR and IF studies showed a significant decrease in expression of profibrotic markers, including α‐smooth muscle actin (α‐SMA; p < .0001), fibronectin (FN; p = .0002), tenascin C (TNC; p < .0001), Collagen I (Col‐I; p < .0001), Collagen IIIA1 (Co‐IIIA1; p < .0001), and Collagen IV (Co‐lV; p < .0001).An ophthalmic formulation consisting of lisinopril and fasudil may offer a safe and effective method to treat canine corneal fibrosis. Additional studies evaluating safety and efficacy of this formulation in vivo are warranted. [ABSTRACT FROM AUTHOR]

Corneal disease remains a leading cause of impaired vision world-wide, and advancements in gene therapy continue to develop with promising success to prevent, treat and cure blindness. Ideally, gene therapy requires a vector and gene delivery method that targets treatment of specific cells or tissues and results in a safe and non-immunogenic response. The cornea is a model tissue for gene therapy due to its ease of clinician access and immune-privileged state. Improvements in the past 5–10 years have begun to revolutionize the approach to gene therapy in the cornea with a focus on adeno-associated virus and nanoparticle delivery of single and combination gene therapies. In addition, the potential applications of gene editing (zinc finger nucleases [ZNFs], transcription activator-like effector nucleases [TALENs], Clustered Regularly Interspaced Short Palindromic Repeats/Associated Systems [CRISPR/Cas9]) are rapidly expanding. This review focuses on recent developments in gene therapy for corneal diseases, including promising multiple gene therapy, while outlining a practical approach to the development of such therapies and potential impediments to successful delivery of genes to the cornea. • Bilateral corneal disorders are a leading cause of blindness worldwide. • Gene therapy is proving a safe, rapid and long-term solution to corneal disorders. • Cornea is an ideal tissue for gene therapy application due to access and monitoring. • rAAV and nanocarriers allow a wide scope of options for gene therapy development. • Multiple gene therapy in preclinical animal models have safe and effective results. [ABSTRACT FROM AUTHOR]

Loss of corneal transparency after trauma, injury or infection results in blindness. Compromised wound healing because of excessive production of myofibroblasts has been identified a major mechanism for the loss of corneal transparency. We tested a hypothesis that de‐differentiation of corneal myofibroblast into precursor corneal stromal fibroblast by silencing of MyoD (basic helix‐loop‐helix) gene will restore corneal transparency by inhibiting alpha smooth muscle actin (αSMA) transcriptional machinery responsible for myofibroblast production. To test the hypothesis, donor human corneas, human corneal stromal fibroblasts (CSFs) and myofibroblasts (CMFs), MyoD/scrambled‐RNAi plasmids, nanoparticles, immunofluorescence, western blotting, qPCR, 2D/3D confocal microscopy, and slot blots were used. The fibrotic human corneas showed significantly greater MyoD expression compared to the normal human cornea (p < 0.05). Likewise, CMFs showed markedly increased MyoD compared to the CSFs in vitro (p < 0.01). The MyoD‐RNAi or scrambled‐RNAi delivery into CSFs and CMFs did not alter their appearance, viability, or proliferation in normal culture conditions. MyoD‐RNAi delivery into CMFs led to phenotype similar to CSFs in a time‐dependent manner. Furthermore, MyoD‐RNAi silencing in CMFs demonstrated significantly deceased αSMA mRNA and protein levels in a time‐dependent manner (p < 0.001). MyoD gene silencing is a novel approach for the restoration of corneal transparency by promoting de‐differentiation of opaque corneal myofibroblasts to transparent corneal keratocytes. [ABSTRACT FROM AUTHOR]

Microbial keratitis is devastating corneal morbidity with a variable spectrum of clinical manifestations depending on the infective etiology. Irrespective of the varied presentation delayed treatment can lead to severe visual impairment resulting from corneal ulceration, possible perforation, and subsequent scarring. Corticosteroids with a potent anti-inflammatory activity reduce host inflammation, thus minimizing resultant scarring while improving ocular symptoms. These potential effects of corticosteroids have been applied widely to treat various corneal diseases ranging from vernal keratoconjunctivitis to dry eye disease. However, antimicrobial therapy remains the mainstay of treatment in microbial keratitis, whereas the use of adjunctive topical corticosteroid therapy remains a matter of debate. Understandably, the use of topical corticosteroids is a double-edged sword with pros and cons in the treatment of microbial keratitis. Herein we review the rationale for and against the use and safety of topical corticosteroids in the treatment of infective keratitis. Important considerations, including type, dose, efficacy, the timing of initiation of corticosteroids, use of concomitant antimicrobial agents, and duration of corticosteroid therapy while prescribing corticosteroids for microbial keratitis, have been discussed. This review intends to provide new insights into the therapeutic utility of steroids as adjunctive treatment of corneal ulcer. [ABSTRACT FROM AUTHOR]

Retinal diseases are one of the leading causes of blindness globally. The mainstay treatments for these blinding diseases are laser photocoagulation, vitrectomy, and repeated intravitreal injections of anti-vascular endothelial growth factor (VEGF) or steroids. Unfortunately, these therapies are associated with ocular complications like inflammation, elevated intraocular pressure, retinal detachment, endophthalmitis, and vitreous hemorrhage. Recent advances in nanomedicine seek to curtail these limitations, overcoming ocular barriers by developing non-invasive or minimally invasive delivery modalities. These modalities include delivering therapeutics to specific cellular targets in the retina, providing sustained delivery of drugs to avoid repeated intravitreal injections, and acting as a scaffold for neural tissue regeneration. These next-generation nanomedicine approaches could potentially revolutionize the treatment landscape of retinal diseases. This review describes the availability and limitations of current treatment strategies and highlights insights into the advancement of future approaches using next-generation nanomedicines to manage retinal diseases. [ABSTRACT FROM AUTHOR]

Sulfur mustard gas (SM) is a vesicating and alkylating agent used as a chemical weapon in many mass-casualty incidents since World War I. Ocular injuries were reported in >90% of exposed victims. The mechanisms underlying SM-induced blindness remain elusive. This study tested the hypothesis that SM-induced corneal fibrosis occurs due to the generation of myofibroblasts from resident fibroblasts via the SMAD2/3 signaling pathway in rabbit eyes in vivo and primary human corneal fibroblasts (hCSFs) isolated from donor corneas in vitro. Fifty-four New Zealand White Rabbits were divided into three groups (Naïve, Vehicle, SM-Vapor treated). The SM-Vapor group was exposed to SM at 200 mg-min/m3 for 8 min at the MRI Global facility. Rabbit corneas were collected on day 3, day 7, and day 14 for immunohistochemistry, RNA, and protein lysates. SM caused a significant increase in SMAD2/3, pSMAD, and ɑSMA expression on day 3, day 7, and day 14 in rabbit corneas. For mechanistic studies, hCSFs were treated with nitrogen mustard (NM) or NM + SIS3 (SMAD3-specific inhibitor) and collected at 30 m, 8 h, 24 h, 48 h, and 72 h. NM significantly increased TGFβ, pSMAD3, and SMAD2/3 levels. On the contrary, inhibition of SMAD2/3 signaling by SIS3 treatment significantly reduced SMAD2/3, pSMAD3, and ɑSMA expression in hCSFs. We conclude that SMAD2/3 signaling appears to play a vital role in myofibroblast formation in the cornea following mustard gas exposure. [ABSTRACT FROM AUTHOR]

One of the most remarkable advancements in medical treatments of corneal diseases in recent decades has been corneal transplantation. However, corneal transplants, including lamellar strategies, have their own set of challenges, such as graft rejection, delayed graft failure, shortage of donor corneas, repeated treatments, and post-surgical complications. Corneal defects and diseases are one of the leading causes of blindness globally; therefore, there is a need for gene-based interventions that may mitigate some of these challenges and help reduce the burden of blindness. Corneas being immune-advantaged, uniquely avascular, and transparent is ideal for gene therapy approaches. Well-established corneal surgical techniques as well as their ease of accessibility for examination and manipulation makes corneas suitable for in vivo and ex vivo gene therapy. In this review, we focus on the most recent advances in the area of corneal regeneration using gene therapy and on the strategies involved in the development of such therapies. We also discuss the challenges and potential of gene therapy for the treatment of corneal diseases. Additionally, we discuss the translational aspects of gene therapy, including different types of vectors, particularly focusing on recombinant AAV that may help advance targeted therapeutics for corneal defects and diseases. [ABSTRACT FROM AUTHOR]

The cornea and cranial dura mater share sensory innervation. This link raises the possibility that pathological impulses mediated by corneal injury may be transmitted to the cranial dura, trigger dural perivascular/connective tissue nociceptor responses, and induce vascular and stromal alterations affecting dura mater blood and lymphatic vessel functionality. In this study, using a mouse model, we demonstrate for the first time that two weeks after the initial insult, alkaline injury to the cornea leads to remote pathological changes within the coronal suture area of the dura mater. Specifically, we detected significant pro-fibrotic changes in the dural stroma, as well as vascular remodeling characterized by alterations in vascular smooth muscle cell (VSMC) morphology, reduced blood vessel VSMC coverage, endothelial cell expression of the fibroblast specific protein 1, and significant increase in the number of podoplanin-positive lymphatic sprouts. Intriguingly, the deficiency of a major extracellular matrix component, small leucine-rich proteoglycan decorin, modifies both the direction and the extent of these changes. As the dura mater is the most important route for the brain metabolic clearance, these results are of clinical relevance and provide a much-needed link explaining the association between ophthalmic conditions and the development of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Dry eye disease (DED) is a chronic ocular surface disorder, associated with inflammation, which can cause severe morbidity, visual compromise, and loss of quality of life, affecting up to 5–50% of the world population. In DED, ocular surface damage and tear film instability due to abnormal tear secretion lead to ocular surface pain, discomfort, and epithelial barrier disruption. Studies have shown the involvement of autophagy regulation in dry eye disease as a pathogenic mechanism along with the inflammatory response. Autophagy is a self‑degradation pathway in mammalian cells that reduces the excessive inflammation driven by the secretion of inflammatory factors in tears. Specific autophagy modulators are already available for the management of DED currently. However, growing studies on autophagy regulation in DED might further encourage the development of autophagy modulating drugs that reduce the pathological response at the ocular surface. In this review, we summarize the role of autophagy in the pathogenesis of dry eye disease and explore its therapeutic application. [ABSTRACT FROM AUTHOR]

An intracameral (IC) injection directly delivers the drug into the anterior chamber of the eye. This targeted drug delivery technique overcomes the ocular barriers and offers a high therapeutic concentration of medication at the desired site and consequently better clinical outcomes. IC drug delivery is a safe and effective modality with many advantages over topical delivery. These include excellent bioavailability, reduced systemic risk, and minimal ocular toxicity. Agents delivered via IC injection have shown promising results against infection, inflammation, ocular hypertension, and neovascularization. Current literature shows that IC antibiotics, including cefuroxime, vancomycin, and moxifloxacin, are routinely used for prophylaxis of endophthalmitis. Other drugs available for IC use are steroids, anesthetics, mydriatics, miotics, antivascular endothelial growth factor, antiglaucoma, and alkylating agents. Introduction of sustained-release devices containing dexamethasone or Bimatoprost in anterior chamber via IC route has the potential in treating ocular inflammation and raised intraocular pressure. The complications such as hemorrhagic occlusive retinal vasculitis and toxic anterior segment syndrome have been documented with IC prophylaxis but are rare. In this review, we provide an overview of available IC drugs, their pharmacokinetics, the spectrum of activity, dosage and preparation, prophylactic and therapeutic usage, clinical efficacy, and safety profiles. [ABSTRACT FROM AUTHOR]

Placental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids. In this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1. Inhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network. Combined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention. [ABSTRACT FROM AUTHOR]

Epigenetics plays a vital role in corneal health and diseases. Epigenetic changes regulate the expression of genes by altering the accessibility of chromatin via histone modifications, DNA methylation and miRNAs without altering DNA sequence. Ocular trauma and infections are common causes of corneal damage, vision impairment, and mono/bilateral blindness worldwide. Mounting literature shows that epigenetic modifications can modulate corneal clarity, function, and pathogenesis including inflammation, wound healing, fibrosis, and neovascularization. Additionally, epigenetic modifications can be targeted to reverse corneal pathologies and develop interventional therapies. However, current understanding on how epigenetic modifications lead to corneal abnormalities and diseases is limited. This review provides in-depth knowledge and mechanistic understanding of epigenetics alterations in corneal pathogenesis, and information on potential epigenetic targets for treatment of corneal diseases. • Epigenetic changes; methylation, acetylation, phosphorylation, and microRNA regulate gene expression and cellular behaviour. • The cornea, a transparent and avascular tissue, is at risk to damage, risking transparency and vision. • Altered epigenetic contribute to corneal pathologies, including fibrosis, wound healing, keratitis, and OSSN. • Limited research highlights the need to study corneal epigenetics, aiding therapy development through better understanding • Epigenetic modifiers may treat corneal pathologies. This emerging field offers potential for novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

This study analyzed the transcriptional changes in primary human corneal stromal fibroblasts (hCSFs) grown under quiescent (serum-free) and proliferating (serum-supplemented) culture conditions to identify genes, pathways, and protein‒protein interaction networks influencing corneal repair and regeneration. Primary hCSFs were isolated from donor human corneas and maintained in serum-free or serum-laden conditions. RNA was extracted from confluent cultures using Qiagen kit and subjected to RNA sequencing (RNAseq) analysis. Differential gene expression (DGE) and pathway enrichment analyses were conducted using DESeq2 and Gene Set Enrichment Analysis (GSEA), respectively. Protein‒protein interaction (PPI) networks were created exploiting the STRING database and analyzed with Cytoscape and the cytoHubba plugin. RNA-seq revealed 5,181 genes that were significantly differentially expressed/changed among the 18,812 annotated genes (p value ˂0.05). A cutoff value of a log2-fold change of ±1.5 or greater was used to identify 674 significantly upregulated and 771 downregulated genes between quiescent and proliferating hCSFs. Pathway enrichment analysis revealed significant changes in genes linked to cell cycle regulation, inflammatory, and oxidative stress response pathways, such as E2F Targets, G2M Checkpoint, and MYC Targets, TNFA signaling via NF-kB, and oxidative phosphorylation. Protein-protein interaction network analysis highlighted critical hub genes. The FGF22, CD34, ASPN, DPT, LUM, FGF10, PDGFRB, ECM2, DCN, VEGFD, OMD, OGN, ANGPT1, CDH5, and PRELP were upregulated, whereas genes linked to cell cycle regulation and mitotic progression, such as BUB1, TTK, KIF23, KIF11, BUB1B, DLGAP5, NUSAP1, CCNA2, CCNB1, BIRC5, CDK1, KIF20A, AURKB, KIF2C, and CDCA8, were downregulated. The RNA sequences and gene count files have been submitted to the Gene Expression Omnibus (accession # GSE260476). Our study provides a comprehensive information on the transcriptional and molecular changes in hCSFs under quiescent and proliferative conditions and highlights key pathways and hub genes. • Total 5,181 significantly regulated genes between quiescent and proliferating hCSFs, show serum's impact on gene expression • The key enriched pathways in the quiescent-hCSFs are related to cell cycle regulation and stress response • Most enriched pathways include E2F Targets, G2M Checkpoint, MYC Targets, TNFA signaling, and oxidative phosphorylation • The study emphasizes the roles of cell cycle and stress response pathways in quiescent-hCSFs [ABSTRACT FROM AUTHOR]

Purpose: Umbilical cord blood serum (UCBS) is an effective adjunctive treatment along with conventional therapy in ocular surface disorders (OSDs). It aids in rapid ocular surface restoration thereby achieving epithelial integrity, in addition to improvement in subjective and objective parameters. The study aims to compare the efficacy of human umbilical cord blood serum and autologous serum (AS) in treatment of OSD. Methods: A prospective randomized study was conducted on 101 eyes diagnosed with OSD resulting from dry eye disease (DED; n = 40), acute chemical burn (ACB; n = 21), and ocular allergy (OA; n = 40). Randomization was done in Group I, administered with AS, and Group II with UCBS. Outcomes evaluated were visual acuity (VA), eye sensation score (ESS), ocular surface disease index (OSDI), tear break-up time (TBUT), Schirmer's value, Corneal Fluorescein Score, epithelial defect, limbal ischemia, corneal clarity (CC), and improvement in grade of severity. Statistical analysis was done using Wilcoxon signed-rank, Wilcoxon rank sum, Chi-square, and Z-test with a significance level (P ≤ 0.05). Results: In DED, Group II showed significant improvement in VA, ESS, and OSDI by the 7th day, whereas the mean Schirmer, TBUT, and corneal fluorescein staining score improved by 3 months. In ACB, Group II showed improvement in VA, reepithelialization, reduction in limbal ischemia, and CC by 3 months. In OA, Group II showed improvement in ESS by day 7. Conclusion: Human umbilical cord blood serum is more effective than AS in restoring ocular surface. [ABSTRACT FROM AUTHOR]

Purpose: Topical, local anesthetic eye drops in conjunction with antibiotics are commonly used to reduce ocular pain and treat patients in emergency clinics; however, their effects on corneal healing are poorly understood. This study examined whether regular or diluted proparacaine eye drops given in combination with common ophthalmic antibiotics affect corneal wound healing parameters using in vitro and in vivo models. Methods: Primary human corneal fibroblasts generated from donor corneas and New Zealand white rabbits were used. Regular (0.5%) and diluted (0.05%) proparacaine eye drops, twice daily for 3 days, were applied to cultures and rabbit eyes, with or without ophthalmic antibiotics (polymyxin B sulfate and trimethoprim). Trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and scratch wound assays measured cellular viability, proliferation, and migration, respectively, in vitro. Slit lamp biomicroscopy, tonometry, fluorescein eye test, hematoxylin and eosin (H&E) staining, and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence were used for in vivo studies. Results: Both regular and diluted proparacaine affected wound healing response in the cornea in vitro and in vivo in a time-dependent manner. Adjunct antibiotic treatments had additive effects characterized by reduced corneal fibroblast viability, proliferation, and migration in vitro and corneal epithelial recovery in vivo. Regular proparacaine with antibiotics showed most pronounced effects on corneal wound healing parameters, and diluted proparacaine without antibiotics had minimal negative effects in vitro and in vivo. Conclusion: Both methods of regular (0.5%) and diluted (0.05%) proparacaine topical application to the cornea are safe, but impede corneal wound healing in vitro and in vivo. Adjunct antibiotic treatments had additive negative effects on corneal wound repair. [ABSTRACT FROM AUTHOR]

Corneal injury and aberrant wound healing commonly result in corneal fibrosis and subsequent vision loss. Intermediate-conductance calmodulin/calcium-activated K+ channels (KCa3.1) have been shown to promote fibrosis in non-ocular and ocular tissues via upregulation of transforming growth factor beta (TGFβ). TRAM-34 is a selective inhibitor of KCa3.1 and reduces fibrosis by downregulation of TGFβ-induced transdifferentiation of stromal fibroblasts to myofibroblasts. Ascorbic acid has been demonstrated to be effective in promoting corneal re-epithelialization and reduction of neovascularization via anti-VEGF and anti-MMP mechanisms. This study evaluates tolerability and efficacy of a novel combination of TRAM-34 (25μM) and ascorbic acid (10%) topical treatment for corneal fibrosis using an established in vivo rabbit model and conducting clinical eye examinations. Markers of corneal fibrosis were evaluated in all corneas at study endpoint via histopathology, immunofluorescence, and quantitative real-time PCR. The eyedrop treated eyes showed significantly improved clinical outcomes based on modified McDonald Shadduck scores, reduction of clinical haze on Fantes scores, and reduction of central corneal thickness (CCT). At cellular and molecular levels, eyedrop treatment also significantly reduced expression of alpha smooth muscle actin (α-SMA) mRNA and protein, collagen III mRNA, and fibronectin mRNA compared to non-treated eyes. Our study suggests that a tested new bimodal eyedrop is well tolerated and effectively reduces corneal fibrosis/haze in rabbits in vivo. [ABSTRACT FROM AUTHOR]

Inflammatory factors have been considered to contribute to keratoconus (KC) pathogenesis. This study aims to determine the immune cells subsets and soluble inflammatory factor profile on the ocular surface of KC patients. 32 KC subjects (51 eyes) across different grades of severity and 15 healthy controls (23 eyes) were included in the study. Keratometry and pachymetry measurements were recorded. Ocular surface immune cells (collected by ocular surface wash) immunophenotyped using flow cytometry include leukocytes, neutrophils, macrophages, natural killer (NK) cells, pan-T cells, gamma delta T (γδT) cells and NKT cells. Tear fluid collected using Schirmer's strip was used to measure 50 soluble factors by multiplex ELISA. Proportions of activated neutrophils, NK cells and γδT cells were significantly increased in KC patients. Significantly higher levels of tear fluid IL-1β, IL-6, LIF, IL-17A, TNFα, IFNα/β/γ, EPO, TGFβ1, PDGF-BB, sVCAM, sL-selectin, granzyme-B, perforin, MMP2, sFasL and IgE, along with significantly lower levels of IL-1α and IL-9 were observed in KC patients. Alterations observed in few of the immuno-inflammatory parameters correlated with grades of disease, allergy, eye rubbing and keratometry or pachymetry measurements. The observation implies a distinct immuno-inflammatory component in KC pathogenesis and its potential as an additional therapeutic target in KC management. [ABSTRACT FROM AUTHOR]

• Mustard gas is a highly toxic alkylating agent that rapidly penetrates into ocular tissues. • Sulfur mustard gas exposure to eye causes multiple ophthalmic manifestations including acute and chronic vision loss. • MGK involves unique acute, chronic, and delayed ocular manifestations that can persist for months and years post-exposure. Sulfur mustard (SM) is a chemical warfare agent that has been used throughout recent history and remains a threat today. Exposed soldiers and civilians experience a variety of symptoms primarily in the respiratory system, skin, and eyes. The ocular tissues are highly sensitive to damage by SM and undergo unique manifestations of acute, chronic, and delayed complications that can persist for months and years after exposure. The mechanisms of this unique mustard gas keratopathy are still not fully understood and animal models for the study of this disease are discussed. Recent advances in mechanisms of injury are included in this review. Ophthalmic manifestations of SM injury including persistent epithelial defects, limbal stem cell deficiency, corneal neovascularization, dry eye, and corneal opacification have been reported. A wide variety of medical and surgical therapies have been studied and are reviewed here along with potential future therapies. [ABSTRACT FROM AUTHOR]

• Acrolein exposure causes oxidative stress in human corneal stromal fibroblasts. • Oxidative stress leads mitochondrial dysfunction and cause cell death after acrolein exposure. • Glutathione can be a potential target to limit acrolein toxicity in human cornea. Toxic and volatile chemicals are widely used in household products and previously used as warfare agents, causing a public health threat worldwide. This study aimed to evaluate the extent of injury and mechanisms of acrolein toxicity in the cornea. Primary human corneal stromal fibroblasts cultures (hCSFs) from human donor cornea were cultured and exposed to acrolein toxicity with -/+ N-acetylcysteine (NAC) to study the mode of action in the presence of Buthionine sulphoximine (BSO). PrestoBlue and MTT assays were used to optimize acrolein, NAC, and BSO doses for hCSFs. Cell-based assays and qRT-PCR analyses were performed to understand the acrolein toxicity and mechanisms. Acrolein exposure leads to an increased reactive oxygen species (ROS), compromised glutathione (GSH) levels, and mitochondrial dysfunction. The TUNEL and caspase assays showed that acrolein caused cell death in hCSFs. These deleterious effects can be mitigated using NAC in hCSFs, suggesting that GSH can be a potential target for acrolein toxicity in the cornea. [ABSTRACT FROM AUTHOR]

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFβ induced fibrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse effects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fibrotic markers without causing any adverse effect on cellular properties. [ABSTRACT FROM AUTHOR]

Acrolein is a highly reactive and volatile unsaturated aldehyde commonly used for producing scores of commercial products. It has been recognized as a chemical weapon since its use during World War I, and more recently, in Syria. Acrolein exposure causes severe eye, skin, and lung damage in addition to many casualties. In the eye, it causes severe pain, eyelid swelling, corneal burns, and vision impairment. Very little information is available about how acrolein damages the cornea and causes vision loss. At present, the lack of clinically relevant animal models limits evaluation of acrolein toxicity and mechanisms specific to the eye. We aim to standardize the mode of delivery and exposure duration of acrolein, damaging the rabbit eye in vivo as an ocular injury model for studying the toxicity of acrolein and developing medical countermeasures. Rabbit eyes were exposed to two modes of delivery (topical and vapor) for different durations (1–5 minutes). Clinical ophthalmic examinations with a slit lamp, stereomicroscope, fluorescein dye, pachymeter, tonometer, and tearing examinations in live rabbits were performed at various times up to 4 weeks. Corneas were histologically diagnosed for transparency, fibrosis, collagens, and neovascularization. Our study successfully established an in vivo rabbit model for evaluating acrolein toxicity to the eye, accounting for different modes and durations of exposure. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide gas (H2S) is a chemical weapon and a common environmental pollutant. H2S intoxication is lethal to humans and animals. H2S contact to the eye can cause vision loss. However, the molecular mechanisms associated with H2S toxicity to the cornea remain unclear, and no specific therapy exists to mitigate ocular damage from H2S. Here, we report H2S‐induced cytotoxicity and the parameters contributing to the molecular mechanisms associated with corneal toxicity using primary human corneal stromal fibroblasts (hCSFs) in vitro. Sodium hydrosulfide (NaSH) was used as a source of H2S, and the cytotoxicity of H2S was determined by treating hCSF cells with varying concentrations of NaSH (0–10 mM) for 0–72 hours. Changes in cell proliferation, oxidative stress factors, and the expression of inflammatory and fibrotic genes were studied using standard commercial kits and qRT‐PCR. NaSH exposure to hCSFs showed dose‐ and time‐dependent cytotoxicity. The IC50 of NaSH was determined to be 5.35 mM. NaSH 5.35 mM exposure led to significantly decreased cytochrome c oxidase activity, increased ROS production, and increased expression of inflammatory and fibrotic genes in hCSF cells. H2S/NaSH exposure alters normal mitochondrial function, oxidative stress, and inflammatory and fibrotic gene responses in corneal stromal fibroblasts in vitro. [ABSTRACT FROM AUTHOR]

Retinal inflammation is an integral component of many retinal diseases including diabetic retinopathy (DR), age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). Inflammation is commonly initiated and perpetuated by myeloid-derived immune cells. In the retina, microglial cells are resident macrophages with myeloid origins, which acts as the first responders involved in the innate immune system. To understand the disease pathogenesis, the use of isolated retinal cell culture model is vital for the examination of multiple cellular responses to injury or trauma. The pig retina resembles human retina in terms of tissue architecture, vasculature, and topography. Additionally, it is a better model than the rodent retina because of the presence of the pseudomacula. In the present study, we sought to establish and characterize pig retinal primary microglial cell (pMicroglia) culture. We used pig eyes from the local abattoir and optimized pMicroglia cultures using multiple cell culture conditions and methods. The best results were obtained by seeding cells in DMEM-high glucose media for 18 days followed by shaking of the culture plate. The resulting pMicroglia were characterized by cellular morphology, phenotype, and immunostaining with Iba-1, CD68, P2Y12, CD163, CD14, and Isolectin GS-IB4. Generated pMicroglia were found functionally active in phagocytosis assay and responsive to lipopolysaccharides (LPS) in dose-dependent production of IL-1β. Furthermore, they showed increased secretion of pro-inflammatory cytokines with LPS treatment. Thus, we report a novel and reproducible method for the isolation of primary microglial cells from pig eyes, which may be useful for studying retinal diseases. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated the efficiency and potential toxicity of a linear 22-kDa polyethylenimine (PEI)-DNA nanoconstruct for delivering genes to corneal cells and the effects of PEI nitrogen-to-DNA phosphate (N:P) ratio on gene transfer efficiency in vitro and in vivo.Methods: A gel retardation assay, zeta potential measurement, bright-field microscopy, transfection with green fluorescent protein (GFP), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to characterize the physicochemical and biological properties and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assay for cytotoxicity of the linear PEI-DNA nanoconstruct using in vitro cultured primary human corneal fibroblast and in vivo mouse models.Results: Of the several evaluated N:P ratios, the highest gene transfection efficiency achieved without any notable cytotoxicity was observed at an N:P ratio of 30:1 (N:P 30). In vivo gene transfer studies revealed substantial GFP gene delivery into the corneas of mice 3 days after a single 5-min topical application without any significant adverse ocular effects. Slit-lamp biomicroscope ophthalmic examination of the mouse exposed to the linear PEI-DNA nanoconstruct showed no evidence of hyperemia (redness), corneal edema, ocular inflammation, or epiphora (excessive tearing).Conclusions: The 22-kDa linear PEI-DNA nanoconstruct is an efficient and well-tolerated vector for corneal gene therapy in vitro and in vivo and could be used as a platform for developing novel gene-based nanomedicine approaches for corneal diseases. [ABSTRACT FROM AUTHOR]

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo. [ABSTRACT FROM AUTHOR]

Objective To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. Methods Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either Smad2, 3, or 4 or in combination with a plasmid overexpressing Smad7 using Lipofectamine 2000™ or Lipofectamine BLOCK-iTTM. Smad-transfected clones were then exposed to TGF-β1 to induce differentiation to myofibroblasts. Immunofluorescence and qRT-PCR techniques quantified levels of ECF differentiation to ECM by measuring alpha smooth muscle actin, a known marker of ECM transdifferentiation. Results Silencing of individual Smad2, 3, or 4 genes or overexpression of Smad7 showed significant inhibition of ECF transdifferentiation (73-83% reduction). Silencing of Smad2 showed the greatest inhibition of ECF transdifferentiation in (a) and was therefore utilized for the combination gene transfer testing. The combination gene transfer consisting of Smad7 overexpression and Smad2 silencing attenuated ECF differentiation significantly; however, the level was not significant compared to the overexpression of Smad7 individually. Conclusions Using gene transfer technology involving profibrotic Smad silencing, antifibrotic Smad overexpression or its combination is a novel strategy to control TGF-β1-mediated fibrosis in equine fibroblasts. Combination gene therapy was not better than single gene therapy in this study. [ABSTRACT FROM AUTHOR]

Postoperative conjunctival fibrosis is common in patients after glaucoma filtration surgery. The calcium activated potassium (KCa3.1) channel has been shown to inhibit fibrosis in many non-ocular tissues. However, its potential in treating ocular fibrosis remains unknown. We tested the anti-fibrotic potential of TRAM34, a selective blocker of KCa3.1 channel, in treating conjunctival fibrosis. Primary human conjunctival fibroblast (HCF) cultures derived from donor tissues. Myofibroblasts causing conjunctival fibrosis were generated by growing HCFs in the presence of TGFβ1 for 72 h. KCa3.1 mRNA and protein expression in HCF was examined with PCR and western blot. The anti-fibrotic potential of TRAM34 was examined by measuring fibrotic gene expression with quantitative PCR (qPCR), immunofluorescence, and western blotting in HCFs in ± TGFβ1 (5 ng/ml) and TRAM34 (0–25 μM). The cytotoxicity of Tram34 was analyzed with trypan blue assay and its role in Smad signaling was studied with immunofluorescence. Expression of KCa3.1 mRNA and protein was detected in HCFs and TGFβ1 treatment to HCFs significantly increased expression of KCa3.1. TRAM34 treatment attenuated transcription of fibrotic markers, αSMA (p < .001), fibronectin (p < .05), collagen I (p < .001) and collagen IV (p < .001) in TGFβ1-induced HCFs. Further, TRAM34 significantly inhibited TGFβ1-stimulated αSMA protein expression (p < .01) and nuclear translocation of fibrotic Smad2/3 in HCFs and showed no significant cytotoxicity (p < .05). The KCa3.1 potassium channel plays a significant role in the prevention of conjunctival fibrosis and TRAM34 has potential to control post surgical bleb fibrosis in patients. In vivo studies are warranted. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) ocular exposure severely damages the cornea and causes vision impairment. At present, no specific therapy exists to mitigate SM-induced corneal injury and vision loss. This study performed transcriptome profiling of naïve, SM-damaged, and SM-undamaged rabbit corneas using RNA-seq analysis and bioinformatic tools to gain a better mechanistic understanding and develop SM-specific medical countermeasures. The mRNA profiles of rabbit corneas 4 weeks post SM vapor exposure were generated using Illumina-NextSeq deep sequencing (Gene Expression Omnibus accession # GSE127708). The RNA sequences of naïve (n = 4), SM-damaged (n = 5), and SM-undamaged (n = 5) corneas were subjected to differential expression (DE) analysis after quality control profiling with FastQC. DE analysis was performed using HISAT2, StringTie, and DESeq2. The log2(FC)±2 and adjusted p˂0.05 were chosen to identify the most relevant genes. A total of 5930 differentially expressed genes (DEGs) (upregulated: 3196, downregulated: 2734) were found in SM-damaged corneas compared to naïve corneas, whereas SM-undamaged corneas showed 1884 DEGs (upregulated: 1029, downregulated: 855) compared to naïve corneas. DE profiling of SM-damaged corneas to SM-undamaged corneas revealed 985 genes (upregulated: 308, downregulated: 677). The DE profiles were subsequently subjected to signaling pathway enrichment, and protein‒protein interactions (PPIs) were analyzed. Pathway enrichment was performed for the genes associated with cellular apoptosis, death, adhesion, migration, differentiation, proliferation, extracellular matrix, and tumor necrosis factor production. To identify novel targets, we narrowed the pathway analysis to upregulated and downregulated genes associated with cell proliferation and differentiation, and PPI networks were developed. Furthermore, protein targets associated with cell differentiation and proliferation that may play vital roles in corneal fibrosis and wound healing post SM injury were identified. • Transcriptome profiling of rabbit corneas exposed to sulfur mustard advances the mechanistic understanding. • A total of 5930 differentially expressed genes in sulfur mustard-induced corneal injury were identified. • Pathway enrichment analysis identified genes associated with key pathways linked to mustard gas keratopathy. • Study identified 481 upregulated and 439 downregulated genes involved in sulfur mustard induced corneal injury. [ABSTRACT FROM AUTHOR]

Acrolein is a highly reactive volatile toxic chemical that injures the eyes and many organs. It has been used in wars and terrorism for wounding masses on multiple occasions and is readily accessible commercially. Our earlier studies revealed acrolein's toxicity to the cornea and witnessed damage to other ocular tissues. Eyelids play a vital role in keeping eyes mobile, moist, lubricated, and functional utilizing a range of diverse lipids produced by the Meibomian glands located in the upper and lower eyelids. This study sought to investigate acrolein's toxicity to eyelid tissues by studying the expression of inflammatory and lipid markers in rabbit eyes in vivo utilizing our reported vapor-cap model. The study was approved by the institutional animal care and use committees and followed ARVO guidelines. Twelve New Zealand White Rabbits were divided into 3 groups: Naïve (group 1), 1-min acrolein exposure (group 2), or 3-min acrolein exposure (group 3). The toxicological effects of acrolein on ocular health in live animals were monitored with regular clinical eye exams and intraocular pressure measurements and eyelid tissues post-euthanasia were subjected to H&E and Masson's trichrome histology and qRT-PCR analysis. Clinical eye examinations witnessed severely swollen eyelids, abnormal ocular discharge, chemosis, and elevated intraocular pressure (p < 0.001) in acrolein-exposed eyes. Histological studies supported clinical findings and exhibited noticeable changes in eyelid tissue morphology. Gene expression studies exhibited significantly increased expression of inflammatory and lipid mediators (LOX, PAF, Cox-2, and LTB4; p < 0.001) in acrolein-exposed eyelid tissues compared to naïve eyelid tissues. The results suggest that acrolein exposure to the eyes causes acute damage to eyelids by altering inflammatory and lipid mediators in vivo. • Acrolein is a highly toxic volatile chemical. • Acrolein is widely used in manufacturing various commercial products and as a warfare agent. • Ocular exposure of acrolein damages ocular surface tissues and causes vision loss. • The pathophysiology of acrolein-induced vision loss is poorly understood. [ABSTRACT FROM AUTHOR]

Objective To develop an ex vivo equine corneal organ culture model. Specifically, to assess the equine cornea's extracellular matrix and cellularity after 7 days using two different culture techniques: either (i) immersion system or (ii) air/liquid interface system, to determine the best ex vivo equine corneal model. Animals Studied Fourteen healthy equine corneas of various breeds. Procedures Equine corneas with 2 mm of perilimbal sclera were freshly harvested from 7 horses undergoing humane euthanasia. One corneal-scleral ring ( CSR) from each horse was randomly placed in the (i) immersion condition organ culture system ( IC), with the contralateral CSR being placed in the (ii) air/liquid interface organ culture system ( ALC) for 7 days. All corneas were evaluated using serial daily gross photography, histology, qPCR, and TUNEL assay. Results corneal-scleral rings placed in the IC (i) had complete loss of corneal transparency on gross photography by 7 days, showed a significant level of corneal stromal disorganization, significantly increased α- SMA levels on qPCR, and apoptosis on TUNEL assay compared to controls. The ALC (ii) had weak stromal disorganization on histopathologic examination and was not significantly different from normal equine corneal controls on all other evaluated parameters. Conclusions The air-liquid interface organ culture system maintains the equine cornea's extracellular matrix and preserves corneal transparency, while the immersion condition results in near complete degradation of normal equine corneal architecture after 7 days in culture. The air-liquid organ culture is a viable option to maintain a healthy equine cornea in an ex vivo setting for wound healing studies. [ABSTRACT FROM AUTHOR]

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision. [ABSTRACT FROM AUTHOR]

Objective To investigate the efficacy of topical 0.2% hyaluronic acid in canine corneal ulcers in vivo. Procedures Six purpose-bred beagles were randomly assigned into two groups (three dogs/group): group A received experimental product (Optimend™, containing 0.2% hyaluronic acid, KineticVet™); group B received control product (Optimend™ without 0.2% hyaluronic acid and supplemented with carboxymethylcellulose). The clinical scorer was masked to product content and subject assignment. Under sedation and topical anesthesia, 6-mm axial corneal epithelial debridements were performed in the left eye. Wounded corneas received standard ulcer treatment and topical product (group A) or control product (group B) three times a day ( TID) until ulcers were healed. Slit-lamp biomicroscopy was performed 6 h after wounding and then every 12 h; findings were graded according to modified McDonald-Shadduck scoring system; extraocular photography was performed after fluorescein stain application at all examination time points. Images were analyzed using NIH image j software to quantify rate of corneal epithelialization. Gelatin zymography was used to analyze matrix metalloproteinase ( MMP) 2 and 9 protein expression in tears collected at set time points during the study period. Results No statistical differences in clinical ophthalmic examination scores, rate of corneal epithelialization, or MMP2 or MMP9 protein expression were found between groups at any tested time point. Conclusions The application of 0.2% hyaluronic acid to standard ulcer medical management is well tolerated. Topical addition of the viscoelastic did not accelerate corneal wound healing compared to a topical control with similar viscosity in this study. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) is a chemical warfare agent (CWA) that causes severe eye pain, photophobia, excessive lacrimation, corneal and ocular surface defects, and blindness. However, SM's effects on retinal cells are relatively meager. This study investigated the role of SM toxicity on Müller glial cells responsible for cellular architecture, inner blood-retinal barrier maintenance, neurotransmitter recycling, neuronal survival, and retinal homeostasis. Müller glial cells (MIO-M1) were exposed to SM analog, nitrogen mustard (NM), at varying concentrations (50–500 μM) for 3 h, 24 h, and 72 h. Müller cell gliosis was evaluated using morphological, cellular, and biochemical methods. Real-time cellular integrity and morphological evaluation were performed using the xCELLigence real-time monitoring system. Cellular viability and toxicity were measured using TUNEL and PrestoBlue assays. Müller glia hyperactivity was calculated based on glial fibrillary acidic protein (GFAP) and vimentin immunostaining. Intracellular oxidative stress was measured using DCFDA and DHE cell-based assays. Inflammatory markers and antioxidant enzyme levels were determined by quantitative real-time PCR (qRT-PCR). AO/Br and DAPI staining further evaluated DNA damage, apoptosis, necrosis, and cell death. Inflammasome-associated Caspase-1, ASC, and NLRP3 were studied to identify mechanistic insights into NM toxicity in Müller glial cells. The cellular and morphological evaluation revealed the Müller glia hyperactivity after NM exposure in a dose- and time-dependent manner. NM exposure caused significant oxidative stress and enhanced cell death at 72 h. A significant increase in antioxidant indices was observed at the lower concentrations of NM. Mechanistically, we found that NM-treated MIO-M1 cells increased caspase-1 levels that activated NLRP3 inflammasome-induced production of IL-1β and IL-18, and elevated Gasdermin D (GSDMD) expression, a crucial component actuating pyroptosis. In conclusion, NM-induced Müller cell gliosis via increased oxidative stress results in caspase-1-dependent activation of the NLRP3 inflammasome and cell death driven primarily by pyroptosis. • Nitrogen mustard (NM) causes dose- and time-dependent cellular abnormalities in human Müller glia. • NM induced oxidative stress, lipid peroxidation and nitrotyrosine expression, in Müller glia. • NM causes Müller gliosis via caspase-1-dependent NLRP3 inflammasome activation and cell death primarily by pyroptosis. • This study unveils new mechanistic understanding for mustard gas exposure to the retinal Müller glia. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) are transmembrane water channel proteins that regulate the movement of water through the plasma membrane in various tissues including cornea. The cornea is avascular and has specialized microcirculatory mechanisms for homeostasis. AQPs regulate corneal hydration and transparency for normal vision. Currently, there are 13 known isoforms of AQPs that can be subclassified as orthodox AQPs, aquaglyceroporins (AQGPs), or supraquaporins (SAQPs)/unorthodox AQPs. AQPs are implicated in keratocyte function, inflammation, edema, angiogenesis, microvessel proliferation, and the wound-healing process in the cornea. AQPs play an important role in wound healing by facilitating the movement of corneal stromal keratocytes by squeezing through tight stromal matrix and narrow extracellular spaces to the wound site. Deficiency of AQPs can cause reduced concentration of hepatocyte growth factor (HGF) leading to reduced epithelial proliferation, reduced/impaired keratocyte migration, reduced number of keratocytes in the injury site, delayed and abnormal wound healing process. Dysregulated AQPs cause dysfunction in osmolar homeostasis as well as wound healing mechanisms. The cornea is a transparent avascular tissue that constitutes the anterior aspect of the outer covering of the eye and aids in two-thirds of visual light refraction. Being the outermost layer of the eye, the cornea is prone to injury. Of the 13 AQP isoforms, AQP1 is expressed in the stromal keratocytes and endothelial cells, and AQP3 and AQP5 are expressed in epithelial cells in the human cornea. AQPs can facilitate wound healing through aid in cellular migration, proliferation, migration, extracellular matrix (ECM) remodeling and autophagy mechanism. Corneal wound healing post-chemical injury requires an integrative and coordinated activity of the epithelium, stromal keratocytes, endothelium, ECM, and a battery of cytokines and growth factors to restore corneal transparency. If the chemical injury is mild, the cornea will heal with normal clarity, but severe injuries can lead to partial and/or permanent loss of corneal functions. Currently, the role of AQPs in corneal wound healing is poorly understood in the context of chemical injury. This review discusses the current literature and the role of AQPs in corneal homeostasis, wound repair, and potential therapeutic target for acute and chronic corneal injuries. • Aquaporins influence homeostasis and stress responses in ocular tissues after chemical trauma. • Aquaporins modulate cellular migration, proliferation, ER stress, ECM remodeling, and autophagy. • Aquaporin 1,3, 4, 5, 7, and 11 are expressed in the cornea. • Mustard gas exposure to eye affects AQP3 and AQP5 in the cornea. [ABSTRACT FROM AUTHOR]

Pesticide exposure to eyes is a major source of ocular morbidities in adults and children all over the world. Carbofuran (CF), N -methyl carbamate, pesticide is most widely used as an insecticide, nematicide, and acaricide in agriculture, forestry, and gardening. Contact or ingestion of carbofuran causes high morbidity and mortality in humans and pets. Pesticides are absorbed in the eye faster than other organs of the body and damage ocular tissues very quickly. Carbofuran exposure to eye causes blurred vision, pain, loss of coordination, anti-cholinesterase activities, weakness, sweating, nausea and vomiting, abdominal pain, endocrine, reproductive, and cytotoxic effects in humans depending on amount and duration of exposure. Pesticide exposure to eye injures cornea, conjunctiva, lens, retina, and optic nerve and leads to abnormal ocular movement and vision impairment. Additionally, anticholinesterase pesticides like carbofuran are known to cause salivation, lacrimation, urination, and defecation (SLUD). Carbofuran and its two major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) are reversible inhibitors of acetylcholinesterase (AChE) which regulates acetylcholine (ACh), a neurohumoral chemical that plays an important role in corneal wound healing. The corneal epithelium contains high levels of ACh whose accumulation by AChE inhibition after CF exposure overstimulates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Hyper stimulation of mAChRs in the eye causes miosis (excessive constriction of the pupil), dacryorrhea (excessive flow of tears), or chromodacryorrhea (red tears). Recent studies reported alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure. This review describes carbofuran toxicity to the eye with special emphasis on corneal morbidities and blindness. • Carbofuran causes multiple corneal/ocular pathologies and blindness in humans. • Carbofuran exposure modulates wound healing and autophagy mechanism in the cornea. • In the eye, miosis, dacryorrhea, chromodacryorrhea, ocular pain and irritation, and blurred vision are post carbofuran exposure. [ABSTRACT FROM AUTHOR]

Objective To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase ( MMP) activity. Methods Cultured canine corneal fibroblasts ( CCF) were incubated in the presence/absence of TGF-β1 (5 ng/mL) and SAHA (2.5 μ m) for 24 h. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase ( MAPK), ERK1/2, and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8, and MMP9 mRNA expressions and MMP2 and MMP9 protein activities, respectively. Results TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho- ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho- JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expressions but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression, but SAHA treatment +/− TGF-β1 significantly increased MMP2 and MMP9 protein expressions. Conclusions The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity. [ABSTRACT FROM AUTHOR]

C-X-C chemokine receptor type 5 (CXCR5) regulates inflammatory responses in ocular and non-ocular tissues. However, its expression and role in the cornea are still unknown. Here, we report the expression of CXCR5 in human cornea in vitro and mouse corneas in vivo , and its functional role in corneal inflammation using C57BL/6J wild-type (CXCR5 +/+) and CXCR5 -deficient (CXCR5 −/−) mice, topical alkali injury, clinical eye imaging, histology, immunofluorescence, PCR, qRT-PCR, and western blotting. Human corneal epithelial cells, stromal fibroblasts, and endothelial cells demonstrated CXCR5 mRNA and protein expression in PCR, and Western blot analyses, respectively. To study the functional role of CXCR5 in vivo , mice were divided into four groups: Group-1 (CXCR5 +/+ alkali injured cornea; n = 30), Group-2 (CXCR5 −/− alkali injured cornea; n = 30), Group-3 (CXCR5 +/+ naïve cornea; n = 30), and Group-4 (CXCR5 −/− naïve cornea; n = 30). Only one eye was wounded with alkali. Clinical corneal evaluation and imaging were performed before and after injury. Mice were euthanized 4 h, 3 days, or 7 days after injury, eyes were excised and used for histology, immunofluorescence, and qRT-PCR. In clinical eye examinations, CXCR5 −/− mouse corneas showed ocular health akin to the naïve corneas. Alkali injured CXCR5 +/+ mouse corneas showed significantly increased mRNA (p < 0.001) and protein (p < 0.01 or p < 0.0001) levels of the CXCR5 compared to the naïve corneas. Likewise, alkali injured CXCR5 −/− mouse corneas showed remarkably amplified inflammation in clinical eye exams in live animals. The histological and molecular analyses of these corneas post euthanasia exhibited markedly augmented inflammatory cells in H&E staining and significant CD11b + cells in immunofluorescence (p < 0.01 or < 0.05); and tumor necrosis factor-alpha (TNFα ; p < 0.05) , cyclooxygenase 2 (COX-2; p < 0.0001) , interleukin (IL)-1β (p < 0.0001) , and IL-6 (p < 0.0001 or < 0.01) mRNA expression compared to the CXCR5 +/+ mouse corneas. Interestingly, CXCR5 −/− alkali injured corneas also showed altered mRNA expression of fibrotic alpha smooth muscle actin (α-SMA; p > 0.05) and angiogenic vascular endothelial growth factor (VEGF; p < 0.01) compared to the CXCR5 +/+ alkali injured corneas. In summary, the CXCR5 gene is expressed in all three major layers of the cornea and appears to influence corneal inflammatory and repair events post-injury in vivo. More studies are warranted to tease the mechanistic role of CXCR5 in corneal inflammation and wound healing. • CXCR5 gene is expressed in human and mouse corneal epithelial, stromal fibroblasts, and endothelial cells. • CXCR5 expression augmented significantly post injury in mouse cornea in vivo. • CXCR5 deficiency led to modified inflammatory, fibrotic, and angiogenic markers in alkali injured cornea in vivo. [ABSTRACT FROM AUTHOR]