Trimebutine prevents corneal inflammation in a rat alkali burn model.

Alkaline burns to the cornea lead to loss of corneal transparency, which is essential for normal vision. We used a rat corneal alkaline burn model to investigate the effect of ophthalmic trimebutine solution on healing wounds caused by alkaline burns. Trimebutine, an inhibitor of the high-mobility group box 1-receptor for advanced glycation end products, when topically applied to the burned cornea, suppressed macrophage infiltration in the early phase and neutrophil infiltration in the late phase at the wound site. It also inhibited neovascularization and myofibroblast development in the late phase. Furthermore, trimebutine effectively inhibited interleukin-1ß expression in the injured cornea. It reduced scar formation by decreasing the expression of type III collagen. These findings suggest that trimebutine may represent a novel therapeutic strategy for corneal wounds, not only through its anti-inflammatory effects but also by preventing neovascularization.

Dexamethasone sodium phosphate loaded nanoparticles for prevention of nitrogen mustard induced corneal injury.

Nitrogen mustard (NM) is a potent vesicating chemical warfare agent that is primarily absorbed through skin, inhalation, or ocular surface. Ocular exposure of NM can cause acute to chronic keratopathy which can eventually lead to blindness. There is a current lack of effective countermeasures against ocular exposure of NM despite their imperative need. Herein, we aim to explore the sustained effect of Dexamethasone sodium phosphate (DSP)-loaded polymeric nanoparticles (PLGA-DSP-NP) following a single subconjunctival injection in the management and prevention of corneal injury progression upon exposure to NM. DSP is an FDA approved corticosteroid with proven anti-inflammatory properties. We formulated PLGA-DSP-NP with zinc chelation ion bridging method using PLGA polymer, with particles of approximately 250 nm and a drug loading of 6.5 wt%. Under in vitro sink conditions, PLGA-DSP-NP exhibited a sustained drug release for two weeks. Notably, in NM injured cornea, a single subconjunctival (SCT) injection of PLGA-DSP-NP outperformed DSP eyedrops (0.1%), DSP solution, placebo NP, and saline, significantly mitigating corneal neovascularization, ulceration, and opacity for the two weeks study period. Through PLGA-DSP-NP injection, sustained DSP release hindered inflammatory cytokine recruitment, angiogenic factors, and endothelial cell proliferation in the cornea. This strategy presents a promising localized corticosteroid delivery system to effectively combat NM-induced corneal injury, offering insights into managing vesicant exposure.

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

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

Treatment of Sulfur Mustard Corneal Injury by Augmenting the DNA Damage Response (DDR): A Novel Approach.

Sulfur mustard (SM) is a highly reactive organic chemical has been used as a chemical warfare agent and terrorist threat since World War I. The cornea is highly sensitive to SM toxicity and exposure to low vapor doses can cause incapacitating acute injuries. Exposure to higher doses can elicit persistent secondary keratopathies that cause reduced quality of life and impaired or lost vision. Despite a century of research, there are no specific treatments for acute or persistent ocular SM injuries. SM cytotoxicity emerges, in part, through DNA alkylation and double-strand breaks (DSBs). Because DSBs can naturally be repaired by DNA damage response pathways with low efficiency, we hypothesized that enhancing the homologous recombination pathway could pose a novel approach to mitigate SM injury. Here, we demonstrate that a dilithium salt of adenosine diphosphoribose (INV-102) increases protein levels of p53 and Sirtuin 6, upregulates transcription of BRCA1/2, enhances γH2AX focus formation, and promotes assembly of repair complexes at DSBs. Based on in vitro evidence showing INV-102 enhancement of DNA damage response through both p53-dependent and p53-independent pathways, we next tested INV-102 in a rabbit preclinical model of corneal injury. In vivo studies demonstrate a marked reduction in the incidence and severity of secondary keratopathies in INV-102-treated eyes compared with vehicle-treated eyes when treatment was started 24 hours after SM vapor exposure. These results suggest DNA repair mechanisms are a viable therapeutic target for SM injury and suggest topical treatment with INV-102 is a promising approach for SM as well as other conditions associated with DSBs. SIGNIFICANCE STATEMENT: Sulfur mustard gas corneal injury currently has no therapeutic treatment. This study aims to show the therapeutic potential of activating the body's natural DNA damage response to activate tissue repair.

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

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

Endogenous TSG-6 modulates corneal inflammation following chemical injury.

PURPOSE: Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) is upregulated in various pathophysiological contexts, where it has a diverse repertoire of immunoregulatory functions. Herein, we investigated the expression and function of TSG-6 during corneal homeostasis and after injury. METHODS: Human corneas, eyeballs from BALB/c (TSG-6+/+), TSG-6+/- and TSG-6-/- mice, human immortalized corneal epithelial cells and murine corneal epithelial progenitor cells were prepared for immunostaining and real time PCR analysis of endogenous expression of TSG-6. Mice were subjected to unilateral corneal debridement or alkali burn (AB) injuries and wound healing assessed over time using fluorescein stain, in vivo confocal microscopy and histology. RESULTS: TSG-6 is endogenously expressed in the human and mouse cornea and established corneal epithelial cell lines and is upregulated after injury. A loss of TSG-6 has no structural and functional effect in the cornea during homeostasis. No differences were noted in the rate of corneal epithelial wound closure between BALB/c, TSG-6+/- and TSG-6-/- mice. TSG-6-/- mice presented decreased inflammatory response within the first 24 h of injury and accelerated corneal wound healing following AB when compared to control mice. CONCLUSION: TSG-6 is endogenously expressed in the cornea and upregulated after injury where it propagates the inflammatory response following chemical injury.

Phenylarsine oxide induced corneal injury involves oxidative stress mediated unfolded protein response and ferroptotic cell death: Amelioration by NAC.

Phenylarsine oxide (PAO), an analog of lewisite, is a highly toxic trivalent arsenical and a potential chemical warfare agent. PAO-induced toxicity has been studied in lung, liver, and skin tissues. Nevertheless, very few studies have been published to comprehend the impact of PAO-induced toxicity on ocular tissues, even though eyes are uniquely vulnerable to injury by vesicants. Notably, arsenical vesicants such as lewisite have been shown to cause edema of eyelids, inflammation, massive corneal necrosis, and blindness. Accordingly, human corneal epithelial cells were used to study the effects of PAO exposure. PAO (100 and 200 nM) induced significant oxidative stress in corneal epithelial cells. Simultaneous treatment with N-acetyl-l-cysteine (NAC), an FDA-approved antioxidant, reversed the PAO-induced toxicity in human corneal epithelial cells. Furthermore, oxidative stress induction by PAO was accompanied by unfolded protein response (UPR) signaling activation and ferroptotic cell death. Further, to validate the findings of our in vitro studies, we optimized injury biomarkers and developed an ex vivo rabbit corneal culture model of PAO exposure. Investigations using PAO in ex vivo rabbit corneas revealed similar results. PAO (5 or 10 µg) for 3, 5, and 10 min caused moderate to extensive corneal epithelial layer degradation and reduced the epithelial layer thickness in a concentration- and time-dependent manner. Similar to human corneal cells, injuries by PAO in ex vivo cultured rabbit corneas were also associated with elevated oxidative stress, UPR signaling, and ferroptosis induction. NAC mitigated PAO-induced corneal injuries in rabbit ex vivo cornea culture as well. The reversal of PAO toxicity upon NAC treatment observed in our studies could be attributed to its antioxidant properties. These findings suggest that PAO exposure can cause significant corneal injury and highlight the need for further mechanistic studies to better understand the pathobiology of different arsenical vesicants, including PAO and lewisite.

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

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

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Subconjunctival Administration of Mesenchymal Stem Cells Alleviates Ocular Inflammation in a Murine Model of Corneal Alkali Burn.

Corneal alkali burns cause extensive damage not only to the cornea but also to the intraocular tissues. As an anti-inflammatory therapy, subconjunctival administration of mesenchymal stem cells (MSCs) for corneal protection after corneal alkali burn has been explored. Little evidence demonstrates the potential of subconjunctival MSCs delivery in protecting the post-burn intraocular tissues. This study aimed to evaluate the therapeutic efficacy of subconjunctival injection of human placental (hP)-MSCs in protecting against ocular destruction after the burn. hP-MSCs were subconjunctivally administered to C57/BL mice after corneal alkali burn. Western blot of iNOS and CD206 was performed to determine the M1 and M2 macrophage infiltration in the cornea. Infiltration of inflammatory cells in the anterior uvea and retina was analyzed by flow cytometry. The TUNEL assay or Western blot of Bax and Bcl2 was used to evaluate the anti-apoptotic effects of MSCs. MSCs could effectively facilitate cornea repair by suppressing inflammatory cytokines IL-1ß, MCP-1, and MMP9, and polarizing CD206 positive M2 macrophages. Anterior uveal and retinal inflammatory cytokines expression and inflammatory cell infiltration were inhibited in the MSC-treated group. Reduced TUNEL positive staining and Bax/Bcl2 ratio indicated the anti-apoptosis of MSCs. MSC-conditioned medium promoted human corneal epithelial cell proliferation and regulated LPS-stimulated inflammation in RAW 264.7 macrophages, confirming the trophic and immunoregulatory effects of MSCs. Our findings demonstrate that subconjunctival administration of MSCs exerted anti-inflammatory and anti-apoptotic effects in the cornea, anterior uvea, and retina after corneal alkali burn. This strategy may provide a new direction for preventing post-event complications after corneal alkali burn.

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

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

Therapeutic Potential of Mesenchymal Stem Cell-Secreted Factors on Delay in Corneal Wound Healing by Nitrogen Mustard.

Ocular surface exposure to nitrogen mustard (NM) leads to severe ocular toxicity which includes the separation of epithelial and stromal layers, loss of endothelial cells, cell death, and severe loss of tissue function. No definitive treatment for mustard gas-induced ocular surface disorders is currently available. The research was conducted to investigate the therapeutic potential of mesenchymal stem cell-conditioned media (MSC-CM) in NM-induced corneal wounds. NM was added to different types of corneal cells, the ocular surface of porcine, and the ocular surface of mice, followed by MSC-CM treatment. NM significantly induced apoptotic cell death, cellular ROS (Reactive oxygen species), and reduced cell viability, metabolic gene expression, and mitochondrial function, and, in turn, delayed wound healing. The application of MSC-CM post NM exposure partially restored mitochondrial function and decreased intracellular ROS generation which promoted cell survival. MSC-CM therapy enhanced wound healing process. MSC-CM inhibited NM-induced apoptotic cell death in murine and porcine corneal tissue. The application of MSC-CM following a chemical insult led to significant improvements in the preservation of corneal structure and wound healing. In vitro, ex vivo, and in vivo results suggest that MSC-CM can potentially provide targeted therapy for the treatment of chemical eye injuries, including mustard gas keratopathy (MGK) which presents with significant loss of vision alongside numerous corneal pathologies.

Limbal stem cell deficiency (LSCD) in rats and mice following whole body exposure to sulfur mustard (SM) vapor.

Ocular injuries following sulfur mustard (SM) exposure are characterized by an acute phase expressed by corneal erosions and inflammation of the anterior segment that after a clinically silent period may be followed by irreversible corneal injuries. The latter includes epithelial defects, chronic inflammation and neovascularization (NV), and were defined in rabbits and in humans as Limbal Stem Cell Deficiency (LSCD), that derived from a delayed loss of corneal epithelial stem cells (ESC), due to secondary processes most likely in the epithelial stem cell (SC) niche. The present study expands our research on SM-induced ocular injury to rodents (rats and mice) following whole body vapor exposure, aiming to define whether the delayed development of LSCD is a general characteristic of SM ocular toxicity. Freely moving rats and mice were exposed to SM vapor (155 µg/l, 10 min). Clinical examination was carried out in rats and included a slit-lamp bio-microscopy, up to 6 months. Eyes were taken for histology at different time points following exposure and evaluation included hematoxylin and eosin (H&E) staining for general morphology, PAS for identification of goblet cells and p63 immunohistochemistry for progenitor epithelial cells. Whole body exposure to SM vapor in rats and mice resulted in acute ocular injury characterized by corneal erosions and ocular inflammation. Following a brief recovery period, 80-90% of the exposed eyes developed corneal NV associated with abnormal corneal epithelium, stromal inflammation and endothelial damage. The late injury was accompanied by migration of conjunctival goblet cells to the cornea and a loss of limbal epithelial progenitor cells, indicating LSCD. The long-term ocular injury shown hereby in rats and mice was consistent with the lesions described in rabbits and in human casualties and demonstrated the general phenomenon of limbal epithelial stem cells deficiency in SM ocular toxicity. The delayed manifestation of this pathology points towards a therapeutic window for the development of medical countermeasures in small animals following exposure in a real life scenario.

Sulfur mustard corneal injury is associated with alterations in the epithelial basement membrane and stromal extracellular matrix.

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8-17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45+ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14-28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.

AIP1 suppresses neovascularization by inhibiting the NOX4-induced NLRP3/NLRP6 imbalance in a murine corneal alkali burn model.

BACKGROUND: Apoptosis signal-regulating kinase 1-interacting protein 1 (AIP1) participates in inflammatory neovascularization induction. NADPH oxidase 4 (NOX4) produces reactive oxygen species (ROS), leading to an imbalance in nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) and NLR family pyrin domain containing 6 (NLRP6) expression. The mechanisms of AIP1, NOX4, ROS and inflammasomes in corneal neovascularization were studied herein. METHODS: C57BL/6 and AIP1-knockout mice were used in this study. The alkali burn procedure was performed on the right eye. Adenovirus encoding AIP1 plus green fluorescence protein (GFP) (Ad-AIP1-GFP) or GFP alone was injected into the right anterior chamber, GLX351322 was applied as a NOX4 inhibitor, and then corneal neovascularization was scored. The expression of related genes was measured by quantitative real-time polymerase chain reaction, western blotting and immunofluorescence staining. 2',7'-Dichlorofluorescin diacetate staining was used to determine the ROS levels. RESULTS: The expression of AIP1 was decreased, while that of cleaved interleukin-1ß (clv-IL-1ß) and vascular endothelial growth factor A (VEGFa) was increased after alkali burn injury. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. NLRP3/NLRP6 expression was imbalanced after alkali burns. GLX351322 reversed the imbalance in NLRP3/NLRP6 by reducing the ROS levels. This treatment also reduced the expression of clv-IL-1ß and VEGFa, suppressing neovascularization. CONCLUSIONS: AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burn injury. Based on the pathogenesis of corneal neovascularization, these findings are expected to provide new therapeutic strategies for patients. Corneal alkali burn injury is a common type of ocular injury that is difficult to treat in the clinic. The cornea is a clear and avascular tissue. Corneal neovascularization after alkali burn injury is a serious complication; it not only seriously affects the patient's vision but also is the main reason for failed corneal transplantation. Corneal neovascularization affects approximately 1.4 million patients a year. We show for the first time that AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burns. The expression of AIP1 was decreased, while that of clv-IL-1ß and VEGFa was increased after alkali burns. We tried to elucidate the specific molecular mechanisms by which AIP1 regulates corneal neovascularization. NOX4 activation was due to decreased AIP1 expression in murine corneas with alkali burns. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. Additionally, NLRP3/NLRP6 expression was unbalanced, with NLRP3 activation and NLRP6 suppression in the corneal alkali burn murine model. Eye drops containing GLX351322, a NOX4 inhibitor, reversed the imbalance in NLRP3/NLRP6 by reducing ROS expression. This treatment also reduced the expression of clv-IL-1ß and VEGFa, reducing neovascularization. Therefore, we provide new gene therapeutic strategies for patients. With the development of neovascularization therapy, we believe that in addition to corneal transplantation, new drug or gene therapies can achieve better results. Video Abstract.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Forkhead Domain Inhibitor-6 Suppresses Corneal Neovascularization and Subsequent Fibrosis After Alkali Burn in Rats.

Purpose: The purpose of this study was to investigate the effects of Forkhead Domain Inhibitor-6 (FDI-6) on regulating inflammatory corneal angiogenesis and subsequent fibrosis induced by alkali burn. Methods: A corneal alkali burn model was established in Sprague Dawley rats using NaOH and the rat eyes were topically treated with FDI-6 (40 µM) or a control vehicle four times daily for 7 days. Corneal neovascularization, inflammation and epithelial defects were observed on days 1, 4, and 7 under a slit lamp microscope after corneal alkali burn. Analysis of angiogenesis-, inflammation-, and fibrosis-related indicators was conducted on day 7. Murine macrophages (RAW264.7 cells) and mouse retinal microvascular endothelial cells (MRMECs) were used to examine the effects of FDI-6 on inflammatory angiogenesis in vitro. Results: Topical delivery of FDI-6 significantly attenuated alkali burn-induced corneal inflammation, neovascularization, and fibrosis. FDI-6 suppressed the expression of angiogenic factors (vascular epidermal growth factor, CD31, matrix metalloproteinase-9, and endothelial NO synthase), fibrotic factors (α-smooth muscle actin and fibronectin), and pro-inflammatory factor interleukin-6 in alkali-injured corneas. FDI-6 downregulated the expression of monocyte chemotactic protein-1, pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-alpha), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3, and vascular endothelial growth factor in RAW264.7 cells and inhibited the proliferation, migration, and tube formation of MRMECs in vitro. Conclusions: FDI-6 can attenuate corneal neovascularization, inflammation, and fibrosis in alkali-injured corneas.

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

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

Drp1-dependent mitochondrial fission mediates corneal injury induced by alkali burn.

Corneal alkali burn, one of the most serious ophthalmic emergencies, is difficult to be cured by conservative treatments. It is well known that oxidative stress, inflammation and neovascularization are the main causes of corneal damage after alkali burn, but its underlying mechanism remains to be elucidated. Here, we reported that the expression and phosphorylation (Ser616) of mitochondrial fission protein Drp1 were up-regulated at day 3 after alkali burn, while mitochondrial fusion protein Mfn2 was down-regulated. The phosphorylation of ERK1/2 in corneas was increased at day 1, 3, 7 and peaked at day 3 after alkali burn. In human corneal epithelial cells (HCE-2), NaOH treatment induced mitochondrial fission, intracellular ROS production and mitochondrial membrane potential disruption, which was prevented by Drp1 inhibitor Mdivi-1. In corneas, Mdivi-1 or knockdown of Drp1 by Lenti-Drp1 shRNA attenuated alkali burn-induced ROS production and phosphorylation of IκBα and p65. In immunofluorescence staining, it was detected that Mdivi-1 also prevented NaOH-induced nuclear translocation of p65 in HCE-2 cells. Moreover, the expression of NADPH oxidase NOX2 and NOX4 in corneas peaked at day 7 after alkali burn. Mdivi-1, Lenti-Drp1 shRNA or the mitochondria-targeted antioxidant mito-TEMPO efficiently alleviated activation of NF-κB, expression of NOX2/4 and inflammatory cytokines including IL-6, IL-1ß and TNF-α in corneas after alkali burn. In pharmacological experiments, both Mdivi-1 and NADPH oxidases inhibitor Apocynin protected the corneas against alkali burn-induced neovascularization. Intriguingly, the combined administration of Mdivi-1 and Apocynin had a synergistic inhibitory effect on corneal neovascularization after alkali burn. Taken together, these results indicate that Drp1-dependent mitochondrial fission is involved in alkali burn-induced corneal injury through regulating oxidative stress, inflammatory responses and corneal neovascularization. This might provide a novel therapeutic target for corneal injury after alkali burn in the future.

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway.

Purpose: Previous work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism. Methods: MNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays. Results: It was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway. Conclusions: This study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.