URP20 improves corneal injury caused by alkali burns combined with pathogenic bacterial infection in rats.

Corneal alkali burns often occur in industrial production and daily life, combined with infection, and may cause severe eye disease. Oxidative stress and neovascularization (NV) are important factors leading to a poor prognosis. URP20 is an antimicrobial peptide that has been proven to treat bacterial keratitis in rats through antibacterial and anti-NV effects. Therefore, in this study, the protective effect and influence mechanism of URP20 were explored in a rat model of alkali burn together with pathogenic bacteria (Staphylococcus aureus and Escherichia coli) infection. In addition, human umbilical vein endothelial cells (HUVECs) and human corneal epithelial cells (HCECs) were selected to verify the effects of URP20 on vascularization and oxidative stress. The results showed that URP20 treatment could protect corneal tissue, reduce corneal turbidity, and reduce the NV pathological score. Furthermore, URP20 significantly inhibited the expression of the vascularization marker proteins VEGFR2 and CD31. URP20 also reduced the migration ability of HUVECs. In terms of oxidative stress, URP20 significantly upregulated SOD and GSH contents in corneal tissue and HCECs (treated with 200 µM H2O2) and promoted the expression of the antioxidant protein Nrf2/HO-1. At the same time, MDA and ROS levels were also inhibited. In conclusion, URP20 could improve corneal injury combined with bacterial infection in rats caused by alkali burns through antibacterial, anti-NV, and antioxidant activities.

Development of dual-functional core-shell electrospun mats with controlled release of anti-inflammatory and anti-bacterial agents for the treatment of corneal alkali burn injuries.

In this study, a novel dual-drug carrier for the co-administration of an anti-inflammatory and antibiotic agent consisting of core-shell nanofibers for the treatment of cornea alkali burns was designed. The core-shell nanofibers were prepared via coaxial electrospinning of curcumin-loaded silk fibroin as the core and vancomycin-loaded chitosan/polyvinyl alcohol (PVA) as the shell. Electron microscopy (SEM and TEM) images confirmed the preparation of smooth, bead-free, and continuous fibers that formed clear core-shell structures. For further studies, nanofiber mats were cross-linked by heat treatment to avoid rapid disintegration in water and improve both mechanical properties and drug release. The release profile of curcumin and vancomycin indicated an initial burst release, continued by the extended release of both drugs within 72 hours. Rabbit corneal cells demonstrated high rates of proliferation when evaluated using a cell metabolism assay. Finally, the therapeutic efficiency of core/shell nanofibers in healing cornea alkali burn was studied by microscopic and macroscopic observation, fluorescence staining, and hematoxylin-eosin assay on rabbit eyes. The anti-inflammatory activity of fabricated fibers was evaluated by enzyme-linked immunosorbent assay and Immunofluorescence analysis. In conclusion, using a robust array of in vitro and in vivo experiments this study demonstrated the ability of the dual-drug carriers to promote corneal re-epithelialization, minimize inflammation, and inhibit corneal neovascularization. Since these parameters are critical to the healing of corneal wounds from alkali burns, we suggest that this discovery represents a promising future therapeutic agent that warrants further study in humans.

Collagen membrane loaded with doxycycline through hydroxypropyl chitosan microspheres for the early reconstruction of alkali-burned cornea.

Corneal alkali burn is one of the most devastating ophthalmic emergencies correlated with remarkable morbidity resulting in severe visual impairment. Appropriate intervention in the acute phase determines the eventual outcome for later corneal restoration treatment. Since the epithelium plays an essential role in inhibiting inflammation and promoting tissue repair, sustained anti-matrix metalloproteinases (MMPs) and pro-epithelialization are the prior remedies during the first week. In this study, a drug-loaded collagen membrane (Dox-HCM/Col) that could be sutured to overlay the burned cornea was developed to accelerate the early reconstruction. Doxycycline (Dox), a specific inhibitor of MMPs, was encapsulated in collagen membrane (Col) through hydroxypropyl chitosan microspheres (HCM) to develop Dox-HCM/Col, affording a preferable pro-epithelialization microenvironment and an in-situ controlled release. Results showed that loading HCM into Col prolonged the release time to 7 days, and Dox-HCM/Col could significantly suppress the expression of MMP-9 and -13 in vitro and in vivo. Furthermore, the membrane accelerated the corneal complete re-epithelialization and promoted early reconstruction within the first week. Overall, Dox-HCM/Col was a promising biomaterial membrane for treating alkali-burned cornea in the early stage, and our attempt may provide a clinically feasible method for the ocular surface reconstruction.

Rapamycin inhibits corneal inflammatory response and neovascularization in a mouse model of corneal alkali burn.

Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.

Upadacitinib inhibits corneal inflammation and neovascularization by suppressing M1 macrophage infiltration in the corneal alkali burn model.

Alkali burn-induced corneal inflammation and subsequent corneal neovascularization (CNV) are major causes of corneal opacity and vision loss. M1 macrophages play a central role in inflammation and CNV. Therefore, modulation of M1 macrophage polarization is a promising strategy for corneal alkali burns. Here, we illustrate the effect and underlying mechanisms of upadacitinib on corneal inflammation and CNV induced by alkali burns in mice. The corneas of BALB/c mice were administered with 1 M NaOH for 30 s and randomly assigned to the vehicle group and the upadacitinib-treated group. Corneal opacity and corneal epithelial defects were assessed clinically. Quantitative real-time PCR (qRT-PCR), immunohistochemistry, and western blot analysis were performed to detect M1 macrophage polarization and CD31+ corneal blood vessels. The results showed that upadacitinib notably decreased corneal opacity, and promoted corneal wound healing. On day 7 and 14 after alkali burns, upadacitinib significantly suppressed CNV. Corneal alkali injury caused M1 macrophage recruitment in the cornea. In contrast to the vehicle, upadacitinib suppressed M1 macrophage infiltration and decreased the mRNA expression levels of inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, and vascular endothelial growth factor A (VEGF-A) in alkali-injured corneas. Moreover, upadacitinib dose-dependently inhibited M1 macrophage polarization by suppressing interferon (IFN)-γ-/lipopolysaccharide-stimulated STAT1 activation in vitro. Our findings reveal that upadacitinib can efficiently alleviate alkali-induced corneal inflammation and neovascularization by inhibiting M1 macrophage infiltration. These data demonstrate that upadacitinib is an effective drug for the treatment of corneal alkali burns.

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model.

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

CXCR3 deletion aggravates corneal neovascularization in a corneal alkali-burn model.

Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.

Effect of Nintedanib Nanothermoreversible Hydrogel on Neovascularization in an Ocular Alkali Burn Rat Model.

PURPOSE: To compare the therapeutic effects of different forms of nintedanib ophthalmic preparations on neovascularization corneal alkali burns in rats. METHODS: Forty rat models of left eye corneal alkali burns were constructed, and the five groups (N = 8) were treated with normal saline, dexamethasone ointment (dexamethasone), 0.2% nintedanib aqueous solution and nintedanib nano thermoreversible hydrogel (NNTH). A slit lamp microscope was used to observe the area of neovascularization. The levels of the inflammatory factors were detected by ELISA. HE staining was performed on the rat corneas. Vascular endothelial growth factor (VEGFA) was detected by immunohistochemistry, and the expression of corneal VEGFA and CD31 was detected by western blotting. An MTT assay was performed to detect the cytotoxicity of nintedanib on human corneal epithelial cells (HCECs) and human umbilical vein vascular endothelial cells (HUVECs). Cell migration was detected by a cell scratch assay, and the proportion of apoptotic cells was detected by Annexin/PI double staining. Immunofluorescence and western blotting were performed to detect the protein expression of VEGFA and CD31. RESULTS: NNTH had a stronger inhibitory effect on corneal neovascularization (CNV) in alkali-burned rats while reducing the level of inflammatory factors. NNTH had a longer drug duration of release than nanoformulations in vitro. Nintedanib at low concentrations (<8 µM) had no significant cytotoxicity to HCECs but significantly induced apoptosis and inhibited the expression of VEGFA and CD31 and the migration of HUVECs. CONCLUSIONS: Nanomorphic thermoreversible hydrogel is superior among the nintedanib ophthalmic preparations, showing better inhibition of CNV in alkali-burned eyeballs and it blocked the migration and proangiogenic ability of HUVECs.

Prophylactic Instillation of Hydrogen-Rich Water Decreases Corneal Inflammation and Promotes Wound Healing by Activating Antioxidant Activity in a Rat Alkali Burn Model.

Many studies have demonstrated the therapeutic effects of hydrogen in pathological conditions such as inflammation; however, little is known about its prophylactic effects. The purpose of this study is to investigate the prophylactic effects of hydrogen-rich water instillation in a rat corneal alkali burn model. Hydrogen-rich water (hydrogen group) or physiological saline (vehicle group) was instilled continuously to the normal rat cornea for 5 min. At 6 h after instillation, the cornea was exposed to alkali. The area of corneal epithelial defect (CED) was measured every 6 h until 24 h after alkali exposure. In addition, at 6 and 24 h after injury, histological and immunohistochemical observations were made and real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to investigate superoxide dismutase enzyme (SOD)1, SOD2, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression. CED at 12 h and the number of inflammatory infiltrating cells at 6 h after injury were significantly smaller in the hydrogen group than the vehicle group. Furthermore, SOD1 expression was significantly higher in the hydrogen group than the vehicle group at both 6 and 24 h, and the number of PGC-1α-positive cells was significantly larger in the hydrogen group than the vehicle group at 6 h after injury. In this model, prophylactic instillation of hydrogen-rich water suppressed alkali burn-induced inflammation, likely by upregulating expression of antioxidants such as SOD1 and PGC-1α. Hydrogen has not only therapeutic potential but also prophylactic effects that may suppress corneal scarring following injury and promote wound healing.

The Effect of Bromfenac Sodium Nanopolymer Used in Anterior Segment of the Eye on Corneal Neovascularization.

This study was to explore the inhibitory effect of bromfenac sodium (BF) / chitosan (CS) nanoparticles (NPs) on corneal neovascularization (CNV). 45 New Zealand white rabbits provided by The First Affiliated Hospital of Jinan University were randomly divided into a control group (group A, n = 15), 0.1% BF aqueous solution treatment group (group B, n = 15), and 0.1% BF/CS-NPs suspension treatment group (group C, n = 15). A rabbit corneal alkali burn model was established. The average particle size of BF/CS-NPs with different BF concentrations was mainly 341.6 ± 12.9 nm - 548.7 ± 15.4 nm; and the Zeta potential distribution was 24.3 ± 2.5 mV - 35.7 ± 4.3 mV. When the initial concentration of BF was 1.5 mg/mL, the maximum drug loading was 57.35 ± 5.26%. The area of CNV in group C was significantly lower than that in groups B and A, and the differences were statistically significant (P < 0.05). At 6, 12, 18, and 24 days after surgery, the mRNA expression levels in cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) gene were compared after standardized by ß-actin; group A had the highest expression level, followed by group B, and group C had the lowest expression level, showing statistically significant differences (P < 0.05). The BF/CS-NPs granules prepared in this study had stable physical and chemical properties and had a good sustained-release effect, and the release duration can be as long as 48 hours. BF/CS-NPs can inhibit the formation of CNV at different time points after alkali burn, and reduce the expression of VEGF and COX-2 in corneal tissue after alkali burn.

Evaluation of clinical and histological effects of KGF-2 and NGF on corneal wound healing in an experimental alkali burn rabbit model.

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-ß (TGF-ß) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-ß was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

Study of the Effectiveness of Drug No. 1 on the Model of Alkaline Eye Burn in Rabbits.

Studies have shown that 0.05 mL/kg of drug No. 1 as 1:15 dilution with sterile saline solution has anti-inflammatory, wound-healing, and angioprotective effects if instilled in both eyes of rabbits twice a day for 30 days after an alkaline burn. A stimulation of reparative processes in the cornea was observed with the test dose of drug No. 1. This was manifested by accelerating the recovery of defects in the anterior epithelium and stroma, reducing the frequency of formation of deep defects and the severity of inflammatory reaction and vascularization, and inhibiting the formation of turbidity of its lower intensity and area. A tendency to restore laminarity of the stroma was determined by the action of drug No. 1 throughout the observation period. This contributed to a decrease in the degree of vascularization and prevented ulceration and perforation of the cornea. By the end of experiment, a restoration of strong epithelial-stromal relationships in the experimental group, compared to the control group, was observed due to formation of normal architectonics of fibrous components of intercellular substance. A more pronounced proliferative activity, with an increase in the layering of limbal epithelial cells, was noted in the limbal zone of the cornea in the experimental group rabbits compared to the control group.

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.

Investigating the Anti-Inflammatory Effects of RCI001 for Treating Ocular Surface Diseases: Insight Into the Mechanism of Action.

The ocular surface is continuously exposed to various environmental factors, and innate and adaptive immunity play crucial roles in ocular surface diseases (OSDs). Previously, we have reported that the topical application of RCI001 affords excellent anti-inflammatory and antioxidant effects in dry eye disease and ocular chemical burn models. In this study, we examined the inhibitory effects of RCI001 on the Rac1 and NLRP3 inflammasomes in vitro and in vivo. Following RCI001 application to RAW264.7 and Swiss 3T3 cells, we measured Rac1 activity using a glutathione-S-transferase (GST) pull-down assay and G-protein activation assay kit. In addition, we quantified the expression of inflammatory cytokines (interleukin [IL]-1ß, IL-6, and tumor necrosis factor [TNF]-α) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells using ELISA and real-time PCR. In the mouse ocular alkali burn model, RCI001 was administered via eye drops (10 mg/mL, twice daily) for 5 days, and 1% prednisolone acetate (PDE) ophthalmic suspension was used as a positive control. Corneal epithelial integrity (on days 0-5) and histological examinations were performed, and transcript and protein levels of Rac1, NLRP3, caspase-1, and IL-1ß were quantified using real-time PCR and western blotting in corneal tissues collected on days 3 and 5. We observed that RCI001 dose-dependently inhibited Rac1 activity and various inflammatory cytokines in LPS-stimulated murine macrophages. Furthermore, RCI001 restored corneal epithelial integrity more rapidly than corticosteroid treatment in chemically injured corneas. Compared to the saline group, activation of Rac1 and the NLRP3 inflammasome/IL-1ß axis was suppressed in the RCI001 group, especially during the early phase of the ocular alkali burn model. Topical RCI001 suppressed the expression of activated Rac1 and inflammatory cytokines in vitro and rapidly restored the injured cornea by inhibiting activation of Rac1 and the NLRP inflammasome/IL-1ß axis in vivo. Accordingly, RCI001 could be a promising therapeutic agent for treating OSDs.

Topical Effects of N-Acetyl Cysteine and Doxycycline on Inflammatory and Angiogenic Factors in the Rat Model of Alkali-Burned Cornea.

The aim of this study was to analyze the single and combined effects of N-acetyl cysteine (NAC) and doxycycline (Dox) on the inflammatory and angiogenic factors in the rat model of alkali-burned cornea. Rats were treated with a single and combined 0.5% NAC and 12.5 µg/mL Dox eye drops and evaluated on days 3, 7, and 28. In the corneas of various groups, the activity of Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes was assessed. The expression of inflammatory factors (TNF-α, Rel-a, and CXCL-1) and angiogenic factors (VEGF-a, MMP2, and MMP9) was measured using real-time polymerase chain reaction. The antioxidant enzyme activities decreased substantially 3 days after injury with sodium hydroxide (NaOH). NAC and combined NAC+ Dox topical treatments increased the SOD enzyme activity on day 28 (P < 0.05). The expression of TNF-α and Rel-a genes following single and combined treatment of NAC and Dox decreased significantly on days 7 and 28 (P < 0.05). The mRNA level of angiogenic factors and corneal neovascularization (CNV) level declined in NaOH-injured rats treated with Dox (P < 0.05). The topical treatment of Dox could attenuate inflammation and CNV complications. However, NAC treatment may not reduce the expression of angiogenic genes.

Comparative Analysis of Matrix-Regenerating Agent and Corneal Cross-Linking in an Experimental Alkali Burn Rabbit Model.

PURPOSE: This study aimed to investigate the clinical and histopathological effects of corneal cross-linking (CXL) and matrix-regenerating agent (RGTA) treatments after corneal alkali burn. MATERIALS AND METHODS: Twenty-four alkali-burned corneas from 24 rabbits were divided into three groups: control, CXL, and RGTA. All animals were investigated for epithelial healing, opacification, ulceration, and neovascularization at days 1, 7, 14, and 21 after the alkali burn. Corneas were excised and sent for histological examination on day 21. RESULTS: One animal each from the CXL and control groups exhibited moderate ulceration, while no ulceration was observed in the RGTA group. No significant difference was observed among the groups in corneal thickness or corneal opacity measurements at the final visit (p = .058 and p = .544, respectively). Both RGTA and CXL treatments were effective in terms of epithelial healing and neovascularization (p = .023 and p = .03, respectively). On histological examination, the CXL and RGTA groups were more effective in treating epithelial loss, stromal edema, corneal vascularization, and leukocytic infiltration than the control group (p < .05). The immunohistochemical staining scores of the CXL and RGTA groups for caspase-3, vascular endothelial growth factor, and matrix metalloproteinase-9 in the epithelium and stroma were significantly lower than those in the control group (p < .05). In the immunohistochemical examination for inducible nitric oxide synthase, epithelial staining scores were similar among the groups (p > .05). In contrast, the stromal staining scores of the CXL and RGTA groups were lower than those of the control group (p < .05). CONCLUSION: Both CXL and RGTA therapies were effective in reducing anatomical and histopathological complications after corneal alkali burn. Further investigation is needed to determine the optimal timing, duration, and dosage of these treatments.

Daphnetin inhibits corneal inflammation and neovascularization on a mouse model of corneal alkali burn.

Alkali burn is a significant contributor to corneal injury. Alkali burn-induced corneal inflammation often causes vision loss due to corneal neovascularization. Daphnetin (DAP) has been studied for its anti-inflammatory and antiangiogenic properties with encouraging results. Driven by those encouraging results, we sought to explore the effects of DAP in treating alkali burn-induced corneal inflammation and neovascularization and its mechanism of action. We found that the angiogenesis processes of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor A (VEGF-A) were primarily attenuated by treatment with DAP, including proliferation, migration, and tube formation. Treatment of DAP significantly suppressed the VEGF-A-induced protein expression of VEGF receptor2 (VEGFR2), as well as the activation of downstream signal transducer and activator of transcription 3 (STAT3), AKT, and extracellular signal-regulated kinase (ERK) signaling. In the mouse corneal alkali burn model, the inflammatory cell infiltrations and neovascularization in the cornea caused by alkali burn were inhibited by 10 µM DAP eye drops. Alkali burn-induced corneal protein expression of VEGF-A, VEGFR2, phosphorylated (p-)STAT3, p-AKT, and p-ERK in corneal tissue were reduced mainly by DAP. Moreover, the upregulation of inflammatory caused by alkali burn in the pathological process was significantly neutralized by DAP. Mechanistically, the inflammatory response could be alleviated by DAP in the way of inhibiting the expression levels of TLR4, p-NF-κB, NLRP3, ASC, Cleaved-caspase-1 (p20), mature-IL-1ß (p17), and N-GSDM. In conclusion, our findings confirmed that the corneal inflammation and neovascularization caused by alkali burn could be inhibited by DAP in vitro and in vivo, elucidating the underlying mechanisms of its protective effects. DAP may have tremendous therapeutic potential for the treatment of corneal alkali burn.

LRG1 facilitates corneal fibrotic response by inducing neutrophil chemotaxis via Stat3 signaling in alkali-burned mouse corneas.

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-ß (TGF-ß) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.

Effect of supersaturated oxygen emulsion treatment on chloropicrin-induced chemical injury in ex vivo rabbit cornea.

With a possibility for the use of chemical weapons in battlefield or in terrorist activities, effective therapies against the devastating ocular injuries, from their exposure, are needed. Oxygen plays a vital role in ocular tissue preservation and wound repair. We tested the efficacy of supersaturated oxygen emulsion (SSOE) in reducing ex vivo corneal and keratocyte injury from chloropicrin (CP). CP, currently used as a pesticide, is a chemical threat agent like the vesicating mustard agents and causes severe corneal injury. Since our previous study in human corneal epithelial cells showed the treatment potential of SSOE (55 %), we further tested its efficacy in an ex vivo CP-induced rabbit corneal injury model. Corneas were exposed to CP (700 nmol) for 2 h, washed and cultured with or without SSOE for 24 h or 96 h. At 96 h post CP exposure, SSOE treatment presented a healing tendency of the corneal epithelial layer, and abrogated the CP-induced epithelial apoptotic cell death. SSOE treatment also reduced the CP induced DNA damage (H2A.X phosphorylation) and inflammatory markers (e.g. MMP9, IL-21, MIP-1ß, TNFα). Further examination of the treatment efficacy of SSOE alone or in combination with other therapies in in vivo cornea injury models for CP and vesicants, is warranted.