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Disorders of the transparent cornea affect millions of people worldwide. However, how to maintain and/or regenerate this organ remains unclear. Here, we show that Rela (encoding a canonical NF-κB subunit) ablation in K14 + corneal epithelial stem cells not only disrupts corneal regeneration but also results in age-dependent epithelial deterioration, which triggers aberrant wound-healing processes including stromal remodeling, neovascularization, epithelial metaplasia, and plaque formation at the central cornea. These anomalies are largely recapitulated in normal mice that age naturally. Mechanistically, Rela deletion suppresses expression of Aldh1a1, an enzyme required for retinoic acid synthesis from vitamin A. Retinoic acid administration blocks development of ocular anomalies in Krt14-Cre; Rela f/f mice and naturally aged mice. Moreover, epithelial metaplasia and plaque formation are preventable by inhibition of angiogenesis. This study thus uncovers the major mechanisms governing corneal maintenance, regeneration, and aging and identifies the NF-κB-retinoic acid pathway as a therapeutic target for corneal disorders., Competing Interests: QY, SB, GM, PZ, BL, JL No competing interests declared, (© 2021, Yu et al.)

Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 μL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45 + immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45 + cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: The aim was to evaluate the ability of gelatin methacryloyl (GelMA) hydrogel eye pads loaded with amniotic extract to prevent symblepharon in rabbits., Materials and Methods: Forty-eight rabbits were divided into 3 groups. After ocular alkali burn, Group A (n=16) was treated with amniotic extract-loaded hydrogel eye pads placed in the conjunctival sac, Group B (n=16) was treated with amniotic membrane transplantation, and Group C (n=16) received no treatment. At 1, 2, 3, and 4 weeks post-injury, 4 rabbits from each group were selected to evaluate for symblepharon, determine epithelial healing rate and corneal neovascularization, conduct histopathology, and to quantify the expression of TGF-β1., Results: At 1 week post-injury, the epithelial healing rate in Groups A and B was higher than Group C (p=0.002, 0.001, respectively). At 2 weeks, corneal neovascularization in Group B was less than Group C (p=0.004). At 3 and 4 weeks, no symblepharon has been found in Group A, but it was found in some eyes in Group B and C (p=0.009, 0.013). Further, the expression of TGF-β1 in Group A was lower than in Group B and C (p<0.001). H&E staining showed that the controls in Group C had more edema and inflammatory cell infiltration in the first 2 weeks, relative to Groups A and B. At 4 weeks, Masson's Trichrome staining showed that fibers were most regularly aligned in Group A and that immuno-histochemical staining found that proliferating cell nuclear antigen was highest expressed in Group C., Conclusions: Treatment with GelMA hydrogel eye pads loaded with amniotic extract shortly after chemical injury prevented symblepharon in rabbits.

Purpose: To compare the therapeutic effects of human derivatives in a mouse alkali burn model. Methods: The right eyes of mice were injured using NaOH. After alkali injury, one of the following agents was topically administered for 7 d: human amniotic membrane (hAM) suspension, human umbilical cord serum (hUCS), and human peripheral blood serum (hPBS), or saline. The epithelial defect areas on days 1, 2, and 3 degrees of opacity on days 2, 3, and 7, and corneal neovascularization (NV) areas on day 7 were evaluated. Histologic examination and mRNA expression levels of tumour necrosis factor (TNF)-α, interleukin (IL)-6, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 were also evaluated on day 7. Results: The epithelial defect areas in the hUCS group were smaller than those in the control and hPBS groups on day 3 ( p  < .05, respectively). The epithelial defect areas in the hAM suspension group showed smaller than those in the control and hPBS groups on days 1 and 2 ( p  < .05, respectively). The degrees of opacity were lower in all treatment groups than that of the saline control group on day 7 ( p  < .05, respectively). Corneal NV areas were not different among groups on day 7 ( p =  0.20). The expression levels of TNF-α, IL-6, MMP-8, and MMP-9 mRNA and the infiltration of the inflammatory cells in all treatment groups were lesser than those in the control group on day 7 ( p<  .05, respectively). Conclusions: All treatments reduced inflammatory reactions and corneal opacity development. Corneal reepithelialization was faster in the hUCS group.

Purpose: To investigate the effect of Hypericum perforatum on corneal alkali burn. Methods: We studied 45 250 g weighing, 4 months old Wistar albino rats. Alkaline burns were performed in the corneas of all experimental animals with 2 mol/L NaOH after general anaesthesia. Rats were divided into five groups according to the subsequent process applied to them: group 1 was the topical Hypericum perforatum group, group 2 was the topical pure olive oil group, group 3 was the oral Hypericum perforatum group, group 4 was the oral pure olive oil group, and group 5 was the control untreated group. Rats were sacrificed under general anaesthesia on the 14 day. The rate of corneal inflammation, neovascularization, fibroblastic activity, and cluster of differentiation 31 (CD31) staining was investigated. Result: There were 45 rats at the beginning of the study. One rat in groups 1, 2, and 3 died during the study; therefore, 42 rats could be evaluated. There were 8 rats in group 1, 8 rats in group 2, 8 rats in group 3, and 9 rats in group 4. We found less corneal neovascularization (CNV), inflammation, and fibroblastic activity in group 1 and group 2 than in the other groups ( p  ˂ 0.001 for all parameters). CNV, inflammation, fibroblastic activity, and CD31 staining rates were lower in group 1 than in group 2 ( p  ˂ 0.001 for all parameters). There was no difference between groups 3, 4, and 5 (respectively, p  = 0.436, 0.634, and 0.750). Conclusions: We found that both topical Hypericum perforatum oily extract and olive oil have anti-inflammatory, anti-angiogenic, and anti-fibroblastic effects when applied after corneal alkali burns in rat corneas. Further studies should be conducted in this field.

Limbal stem cell deficiency (LSCD) is one of the serious cause of visual impairment and blindness with loss of corneal clarity and vascularization. Factors such as ocular burns (acids, lime, thermal), genetic disorders or infections results in the loss of limbal stem cells leading to LSCD. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of the present study was to validate small and large animal models of LSCD by immunohistochemcal, clinical and histopathological comparison with human. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n = 12) and New Zealand white rabbits (r, n = 12) as per the standard existing protocol. Human corneal specimens (h, n = 12) were obtained from tissue bank who had chemical burn-induced LSCD. All samples were either paraffin embedded or frozen in cryogenic medium and the sections were processed for Hematoxylin-Eosin and Periodic Acid-Schiff staining to analyse the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) and epidermal (CK10+) epithelial phenotype. Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p = 0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p = 0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p = 0.18), intra epithelial edema (h:42%, m:33%, r:100%, p = 0.02), stromal inflammation (h:100%, m:67%, r:67%, p = 0.01) and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19 + and/or CK10+, CK3-) in 92% of human and 50% of animal specimens. While partial LSCD (CK19 + and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens. Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma , on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1β) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2 , Il-1β , and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2 , Il-1β , and Vegf . These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.

Chemical burns are a major cause of corneal haze and blindness. Corticosteroids are commonly used after corneal burns to attenuate the severity of the inflammation-related fibrosis. While research efforts have been aimed toward application of novel therapeutics. In the current study, a novel drug delivery system based nanostructured lipid carriers (NLCs) were designed to treat corneal alkaline burn injury. Rapamycin, a potent inhibitor of mammalian target of rapamycin pathway, was loaded in NLCs (rapa-NLCs), and the NLCs were characterized. Cell viability assay, cellular uptake of NLCs, and in vitro evaluation of the fibrotic/angiogenic genes suppression by rapa-NLCs were carried out on human isolated corneal fibroblasts. Immunohistochemistry (IHC) assays were also performed after treatment of murine model of corneal alkaline burn with rapa-NLCs. According to the results, rapamycin was efficiently loaded in NLCs. NLCs could enhance coumarin-6 fibroblast uptake by 1.5 times. Rapa-NLCs efficiently downregulated platelet-derived growth factor and transforming growth factor beta genes in vitro. Furthermore, proliferation of fibroblasts, a major cause of corneal haze after injury, reduced. IHC staining of treated cornea with alpha-smooth muscle actin and CD34 + antibodies showed efficient prevention of myofibroblasts differentiation and angiogenesis, respectively. In conclusion, ocular delivery of rapamycin using NLCs after corneal injury may be considered as a promising antifibrotic/angiogenic treatment approach to preserve patient eyesight., (© 2018 Wiley Periodicals, Inc.)

Background: Since recent reports have shown that (-)-Epigallocatechin-3-gallate (EGCG) could be used for treating proliferative and inflammatory disorders, we explored its use for the management of corneal chemical burns., Materials and Methods: Initially, EGCG was assayed on the rabbit corneal epithelial cell line RCE1(5T5) to establish the best testing conditions, and to avoid unwanted outcomes in the experimental animals. Then, we studied its effects on cell proliferation, cell cycle progression and cell differentiation. Afterwards, we instilled EGCG in experimental grade II corneal alkali burns in mice, three times a day up to 21days, and evaluated by slit lamp examination and histological sections of corneal epithelial, corneal endothelial and stromal edema, as well as the presence of inflammatory cells and neovascularization., Results: EGCG reduced cell growth and led to a decline in the proportion of proliferative cells in a concentration dependent manner. At 10μM, EGCG promoted cell differentiation, an effect not related with apoptosis or cytotoxicity. When 10μM EGCG was instilled in corneal alkali burns in mice three times a day up to 21days, EGCG significantly reduced corneal opacity and neovascularization. The improved clinical appearance of the cornea was associated to a controlled epithelial growth; epithelial morphology was similar to that observed in normal epithelium and contrasted with the hyperproliferative, desquamating epithelium observed in control burn wounds. EGCG reduced corneal, stromal and endothelial edema, and wound inflammation., Conclusion: This work constitutes the first evidence for the use of EGCG in the acute phase of a corneal alkali burn, representing a possible novel alternative to improve patient outcomes as an add-on therapy., (Copyright © 2018 Elsevier Ltd and ISBI. All rights reserved.)

Expression of hypoxia-inducible factor (HIF) 1α has been observed in corneal neovascularization (CNV). Vascular endothelial growth factor (VEGF), one of the most well-known angiogenic factors in CNV, is under the regulation of HIF-1. The present study aims to investigate the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV in rabbits. The models of rabbits in corneal alkali burn were established. SiRNA recombinant adenovirus was used to explore the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV. CNV area and ultrastructure of cornea were observed. The expression of VEGF and HIF-1α was detected. CNV was observed in rabbits following alkali burn. In addition, overexpressed VEGF and HIF-1α was also observed in rabbits following alkali burn. Then, silencing HIF-1α or silencing VEGF decreased area of CNV, inhibited neovascularization and improved pathological changes, while double-target interference for VEGF and HIF-1α decreased area of CNV inhibited neovascularization, and improved pathological changes to a greater extent. Our study provides evidences emphasizing the distinct notion that VEGF and HIF-1α play the contributory role in alkali burn-induced CNV as a result of double-target interference for VEGF and HIF-1α inhibiting CNV in rabbits following corneal alkali burn., (© 2019 The Author(s).)

Purpose: To evaluate the therapeutic effects of topical 8-oxo-2'-deoxyguanosine (8-oxo-dG) on experimental ocular chemical injury models., Methods: We created ocular chemical injury models with 8-week-old BALB/c mice (n = 70) by applying 100% ethanol; the mice were then treated with 8-oxo-dG eye drops 10 and 5 mg/mL and phosphate-buffered saline (PBS) twice daily. After 7 days, clinical findings such as corneal integrity, clarity, and neovascularization were assessed. Histology, immunohistochemistry findings, and inflammatory cytokine levels using real-time polymerase chain reactions in the corneas of the mice were also analyzed., Results: Topical application of 8-oxo-dG eye drops resulted in a significant improvement of epithelial defects and clarity, dose dependently (each P < 0.001). Inflammatory cell infiltration and corneal stromal edema were also decreased in the 8-oxo-dG-treated mice compared with PBS-treated controls, based on hematoxylin and eosin staining. The expressions of F4/80 and neutrophil elastase-positive inflammatory cells and IL-1 and TNF-α cytokine levels were significantly reduced in the 8-oxo-dG group compared with the PBS group (each P < 0.01)., Conclusions: Topical 8-oxo-dG application showed an excellent therapeutic effect in ocular chemical injury models by suppressing inflammation.

The aim of the present study was to investigate the effect of microRNA 146a (miR146a) on promoting the repair of corneal alkali burn with bone marrow mesenchymal stem cells (MSCs). A total of 24 Sprague‑Dawley female rats were divided into a normal group (Control), a normal MSC treatment group (Normal MSCs), an miR146a knockout MSC treatment group (miR146a‑low MSCs) and an miR146a high‑expression MSC treatment group (miR146a‑high MSCs) according to the random number table. Quantitative polymerase chain reaction was used to evaluate the expression levels of miR146a. MTT assay was performed to measure the cell viability of mesenchymal stem cells (MSCs) and apoptosis was measured by flow cytometry. The expression levels of p65 nuclear factor (NF)‑κB, proliferating cell nuclear antigen (PCNA) and Fas proteins were analyzed by western blotting. MSCs were tested for the secretion levels of vascular endothelial growth factor (VEGF), CD45, interferon (IFN)‑γ and interleukin (IL)‑10 by ELISA. The miR146a‑high MSCs improved cell viability of MSCs and inhibited apoptosis of MSCs following alkali burn. miR146a‑high MSCs decreased the expression levels of p65NF‑κB and PCNA, and enhanced the expression level of Fas. Furthermore, miR146a‑high MSCs improved the cornea opacity and enhanced the inhibition of neovascularization in the rats following alkali burn. miR146a‑high MSCs inhibit the expression of VEGF, CD45, IFN‑γ, while enhanced the expression of IL‑10. Therefore, miR146a promotes the repair of corneal alkali burn in rats treated with MSCs.

Purpose: Our previous study demonstrated that Mab2F1, a murine monoclonal antibody blocking the Wnt/β-catenin signaling pathway, has beneficial effects on experimental diabetic retinopathy and choroidal neovascularization (NV). The aforementioned antibody has been humanized. This study evaluated effects of the humanized antibody, H1L1, on NV., Methods: H1L1 was evaluated in the alkali burn-induced corneal NV rat model. Rats with corneal NV were injected subconjunctivally with Mab2F1 or H1L1 using non-specific mouse or human IgG as controls. Corneal NV and opacity were evaluated using corneal NV area and inflammatory index. Expression of angiogenic and inflammatory factors and components of the Wnt/β-catenin pathway in both the corneas of the animal model and human corneal epithelial (HCE) cells exposed to Wnt3a conditioned medium (WCM) were determined by Western blotting and a luciferase-based promoter assay. Cytotoxicities of these antibodies were evaluated by MTT assay., Results: H1L1 reduced the area of corneal NV and opacity, similar to Mab2F1. Both Mab2F1 and H1L1 down-regulated the overexpression of angiogenic and inflammatory factors including VEGF, TNF-α and ICAM-1, and blocked the aberrant activation of the Wnt/β-catenin pathway as shown by down-regulation of phosphorylated LRP6, total LRP6 and non-phosphorylated β-catenin in the cornea of the NV model and cultured HCE cells exposed to WCM. Both antibodies also inhibited the transcriptional activity of β-catenin induced by WCM in HCE cells. No toxic effects of the antibodies were observed in cultured HCE cells., Conclusions: H1L1 exhibits anti-angiogenic activities through blocking the Wnt/β-catenin pathway., (Copyright © 2018. Published by Elsevier Inc.)

Purpose: To evaluate the efficacy of ziv-aflibercept as a treatment for established corneal neovascularization (NV) and to compare its efficacy to that of bevacizumab following ocular chemical insult of sulfur mustard (SM) in the rabbit model., Methods: Chemical SM burn was induced in the right eye of NZW rabbits by vapor exposure. Ziv-aflibercept (2 mg) was applied once to neovascularized eyes by subconjunctival injection while subconjunctival bevacizumab (5 mg) was administered twice a week, for 3 weeks. Non-treated exposed eyes served as a control. A clinical follow-up employed by slit-lamp microscope, was performed up to 12 weeks following exposure and digital photographs of the cornea were taken for measurement of blood vessels length using the image analysis software. Eyes were taken for histological evaluation 2, 4 and 8 weeks following treatment for general morphology and for visualization of NV, using H&E and Masson Trichrome stainings, while conjunctival goblet cell density was determined by PAS staining., Results: Corneal NV developed, starting as early as two weeks after exposure. A single subconjunctival treatment of ziv-aflibercept at 4 weeks post exposure, significantly reduced the extent of existing NV already one week following injection, an effect which lasted for at least 8 weeks following treatment, while NV in the non-treated exposed eyes continued to advance. The extensive reduction in corneal NV in the ziv-aflibercept treated group was confirmed by histological evaluation. Bevacizumab multiple treatment showed a benefit in NV reduction, but to a lesser extent compared to the ziv-aflibercept treatment. Finally, ziv-aflibercept increased the density of conjunctival goblet cells as compared to the exposed non-treated group., Conclusions: Subconjunctival ziv-aflibercept single treatment presented a highly efficient long-term therapeutic benefit in reducing existing corneal NV, following ocular sulfur mustard exposure. These findings show the robust anti-angiogenic efficacy of ziv-aflibercept and demonstrate the advantage of this treatment over the other anti-angiogenic therapies in ameliorating corneal NV and protecting the ocular surface., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: Tetramethylpyrazine (TMP) is the active ingredient extracted from the Chinese herb Chuanxiong. The purpose of our study was to identify the mechanism of therapeutic TMP suppression of pathologic chemokine receptor 4 (CXCR4) transcription., Methods: C57BL/6J mice with alkali-burned corneas were treated with either TMP eye drops (1.5 mg/mL) or PBS. Corneal neovascularization (CNV) was measured and a clinical assessment was made by slit lamp microscopy. Expression of CXCR4 and the transcription factors nuclear respiratory factor-1 (NRF-1), nuclear factor kappa B (NFκB), forkhead box C1, and yin yang 1 were tracked by real-time RT-PCR and immunofluorescence staining of murine corneas. Western blot, real-time PCR, and immunofluorescence evaluated expression of related genes in human umbilical vein endothelial cells (HUVECs) after 200-μmol/L TMP treatment. In addition, plasmid transfection and chromatin immunoprecipitation assays elucidated the relationship among NRF-1, NFκB, and CXCR4., Results: Corneas treated with TMP had smaller areas of neovascularization and scored better in clinical assessments. Injured corneas showed significantly elevated expressions of NRF-1, NFκB, and CXCR4 that were normalized in vivo by TMP treatment. Similarly, in HUVECs in vitro, TMP decreased expression of NRF-1, NFκB, and CXCR4. Overexpression of NFκB or NRF-1 raised the expression of CXCR4 in HUVECs, but not synergistically. Chromatin immunoprecipitation assays detected only NRF-1 bound to the CXCR4 promoter region, suggesting NFκB controls CXCR4 expression by upregulating NRF-1. Together, our data suggest TMP downregulates CXCR4 by repressing NRF-1 expression in CNV, likely indirectly by downregulating NFκB., Conclusions: Our results implicate a novel mechanism wherein TMP inhibits neovascularization via an NFκB/NRF-1/CXCR4 circuit.

Purpose: The aim of this study was to evaluate the inhibitory effect of different concentrations of KH902 eye drops on rabbit corneal neovascularization (CNV) induced by alkali burn., Methods: Forty-eight adult rabbits were randomized into four groups after alkali burning: Group A (2.5 mg/ml), Group B (5 mg/ml), and Group C (10 mg/ml) by different concentrations of KH902 eye drops and Group D by saline solution as control with three times a day for 2 weeks. At days 7, 14, and 28, the anterior segment photographs, confocal microscopy, and histopathology were performed to evaluate corneal opacity, neovascularization, inflammatory cell density, vessel size, and edema. Immunohistochemistry was applied to analyze the vascular endothelial growth factor (VEGF) level., Results: (1) The CNV in the medicine-treated groups showed a reduction without obvious corneal side effects histologically. (2) Compared to the control group, the three medicine-treated groups showed a reduction in the VEGF levels and CNV areas on days 7, 14, and 28 and in the inflammatory cell density on days 14 and 28 (P < 0.01). The difference of inflammatory cell density between the three medicine-treated groups existed on day 14 (P < 0.01). There were differences in the VEGF levels between Groups A, B, and C on days 7, 14, and 28 (P < 0.01), not for Groups B and C on day 28 (P > 0.05)., Conclusion: KH902 eye drops in lower concentration showed an obvious reduction of the CNV growing for rabbit corneal alkali burn without side effects.

Purpose: To characterize the molecular, clinical, and histopathological profiles in the rat cornea after alkali injury over a 21-day period., Methods: Alkali injury was induced in one eye of male Lewis rats. Corneal opacity and corneal neovascularization were assessed daily. Real-time qRT-PCR analysis and immunohistochemical staining were conducted to examine inflammation, neovascularization, and fibrosis., Results: We found that within 2 hours of chemical exposure, corneal opacification rapidly developed with an acute increase in various cytokine expressions, while several cytokines demonstrated a secondary peak by day 7. Early neutrophil infiltration peaked at day 1 post-injury while macrophage infiltration peaked at day 7. Throughout the time course of the study, corneal opacity persisted and neovascularization, lymphangiogenesis, and fibrosis progressed., Conclusions: This study highlights the molecular, clinical, and histopathological changes throughout the progression of alkali injury in the rat cornea. These profiles will assist in the development of new strategies and therapies for ocular alkali injury.

Cornea is an avascular transparent tissue. Ocular trauma caused by a corneal alkali burn induces corneal neovascularization (CNV), inflammation, and fibrosis, leading to vision loss. The purpose of this study was to examine the effects of Zerumbone (ZER) on corneal wound healing caused by alkali burns in mice. CNV was induced by alkali-burn injury in BALB/C female mice. Topical ZER (three times per day, 3μl each time, at concentrations of 5, 15, and 30μM) was applied to treat alkali-burned mouse corneas for 14 consecutive days. Histopathologically, ZER treatment suppressed alkali burn-induced CNV and decreased corneal epithelial defects induced by alkali burns. Corneal tissue treated with ZER showed reduced mRNA levels of pro-angiogenic genes, including vascular endothelial growth factor, matrix metalloproteinase-2 and 9, and pro-fibrotic factors such as alpha smooth muscle actin and transforming growth factor-1 and 2. Immunohistochemical analysis demonstrated that the infiltration of F4/80 and/or CCR2 positive cells was significantly decreased in ZER-treated corneas. ZER markedly inhibited the mRNA and protein levels of monocyte chemoattractant protein-1 (MCP-1) in human corneal fibroblasts and murine peritoneal macrophages. Immunoblot analysis revealed that ZER decreased the activation of signal transducer and activator of transcription 3 (STAT3), with consequent reduction of MCP-1 production by these cells. In conclusion, topical administration of ZER accelerated corneal wound healing by inhibition of STAT3 and MCP-1 production., (Copyright © 2017. Published by Elsevier B.V.)

Objective: The purpose of this study is to explore the inhibitory effects of S100A4 gene silencing on alkali burn-induced corneal neovascularization (CNV) in rabbit models., Methods: Sixty-five rabbits were used to establish alkali-induced CNV models. After the operation, rabbits were given daily antibiotic eye drops and an eye ointment to prevent infection. The models were assigned to either an S100A4 siRNA or an empty vector group. Thirty rabbits were selected as the normal control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the mRNA expression of S100A4 , vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α) in corneal tissues. Immunohistochemistry was used to detect the protein expression of VEGF in corneal tissues, and an enzyme-linked immunosorbent (ELISA) assay was used to detect the protein expression of VEGF and TNF-α in the aqueous humor., Results: The qRT-PCR results showed that S100A4 mRNA expression was lower in the S100A4 siRNA group than in the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. When compared with the empty vector group, the expression of VEGF and TNF-α mRNA was downregulated in the S100A4 siRNA group. The immunohistochemistry results revealed that VEGF protein expression was downregulated in the S100A4 siRNA group when compared to the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. The ELISA results suggest that VEGF and TNF-α protein expression is downregulated in the S100A4 siRNA group in comparison to the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn., Conclusions: These findings indicate that S100A4 gene silencing can inhibit alkali burn-induced CNV in rabbits.

Chemical burns are a major cause of corneal injury. Oxidative stress, inflammatory responses and neovascularization after the chemical burn aggravate corneal damage, and lead to loss of vision. Although NADPH oxidases (Noxs) play a crucial role in the production of reactive oxygen species (ROS), the role of Noxs in chemical burn-induced corneal injury remains to be elucidated. In the present study, the transcription and expression of Noxs in corneas were examined by RT-qPCR, western blot analysis and immunofluorescence staining. It was found that alkali burns markedly upregulated the transcription and expression of Nox2 and Nox4 in human or mouse corneas. The inhibition of Noxs by diphenyleneiodonium (DPI) or apocynin (Apo) effectively attenuated alkali burn-induced ROS production and decreased 3-nitrotyrosine (3-NT) protein levels in the corneas. In addition, Noxs/CD11b double‑immunofluorescence staining indicated that Nox2 and Nox4 were partially co-localized with CD11b. DPI or Apo prevented the infiltration of CD11b-positive inflammatory cells, and inhibited the transcription of inflammatory cytokines following alkali burn-induced corneal injury. In our mouse model of alkali burn-induced corneal injury, corneal neovascularization (CNV) occurred on day 3, and it affected 50% of the whole area of the cornea on day 7, and on day 14, CNV coverage of the cornea reached maximum levels. DPI or Apo effectively attenuated alkali burn‑induced CNV and decreased the mRNA levels of angiogenic factors, including vascular endothelial growth factor (VEGF), VEGF receptors and matrix metalloproteinases (MMPs). Taken together, our data indicate that Noxs play a role in alkali burn-induced corneal injury by regulating oxidative stress, inflammatory responses and CNV, and we thus suggest that Noxs are a potential therapeutic target in the future treatment of chemical-induced corneal injury.

In this study we tried to develop a new approach to suppress inflammation and neovascularization in the alkali-injured rabbit cornea. For this reason Cyclosporine A (CsA)-loaded electrospun nanofibers were transferred onto the ocular surface injured with alkali (0.25 N NaOH). Damaged corneas were divided into the following groups: untreated, treated with CsA eye drops, treated with nanofibers drug-free and treated with CsA-loaded nanofibers. Healthy rabbit corneas served as controls. Drug-free nanofibers and CsA-loaded nanofibers were transferred onto the damaged corneal surface immediately after the injury and sutured to conjunctiva. On day five after the injury the nanofibers were removed. The animals from all groups were sacrificed on day twelve after the injury. The extent of the inflammatory reaction and corneal healing were examined macroscopically, immunohistochemically and biochemically. The central corneal thickness was measured using an ultrasonic pachymeter. When compared with untreated injured corneas, injured corneas treated with drug-free nanofibers or injured corneas treated with CsA eye drops, the number of CD3-positive cells (T lymphocytes) and the production of pro-inflammatory cytokines were strongly reduced in corneas treated with CsA-loaded nanofibers, which was associated with the significantly decreased expression of matrix metalloproteinase 9, inducible nitric oxide synthase, vascular endothelial growth factor and active caspase-3. CsA-loaded nanofibers effectively suppressed corneal inflammation and corneal neovascularization. Central corneal thickness restored to levels before injury only in corneas treated with CsA-loaded nanofibers. Corneal transparency was highly restored in these corneas. It is suggested that the beneficial effect of CsA-loaded nanofibers was associated with the continuous release of CsA from nanofibers and continuous affection of damaged cornea by CsA. The suture of nanofibers to conjunctiva and the closed eyes contributed to beneficial corneal healing. This is in contrast to CsA eye drops, which are quickly washed from the ocular surface and the contact of CsA with the damaged cornea was limited. In conclusion, the approach with CsA-loaded nanofibers could represent an effective alternative mode of therapy for corneal chemical burns., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one method, corneal and limbal epithelia were entirely removed with a 0.5 mm metal burr. In the other, limbus to limbus epithelial removal with the burr was followed by thermal injury to the limbus. These two methods were compared with a previously published one. Unwounded corneas were used as control. The corneas were examined monthly for three months by slit lamp with fluorescein staining. Immunofluorescence staining for cytokeratin 12 and 8 on corneal wholemount and cross sections were performed to determine the phenotype of the epithelium. Mechanical shaving of the epithelium, with or without thermal injury, resulted in a reproducible state of LSCD marked by superficial neovascularization, reduce of keratin 12 expression and presence of goblet cells on the cornea. The phenotype was stable in 100% of the eyes up to at least three months. Thermal injury produced a more severe phenotype with more significant stromal opacification. These corneal injury models may be useful for studying the mechanisms leading to limbal stem cell deficiency., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice., Methods: To observe the effect of fasudil, mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury, CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14., Results: The incidence of CNV was significantly lower after treatment with 100 μM and 300 μM fasudil than with PBS, especially with 100 μM fasudil. Meanwhile, the incidences of corneal epithelial defects was lower (n=15, all p<0.01). After treatment with 100 μM fasudil, the intensity of DHE fluorescence was reduced in the corneal epithelium and stroma than with PBS treatment (n=5, all p<0.01), and the number of filtrated PMNs decreased. There were significant differences between the expressions of VEGF, TNF-a, MMP-8, and MMP-9 in the 100 μM fasudil group and the PBS group (n=8, all p<0.05). The production of HO-1 protein in the 100 μM fasudil group was 1.52±0.34 times more than in the PBS group (n=5 sample, p<0.05)., Conclusions: 100 μM fasudil eye drops administered four times daily can significantly inhibit alkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration, reduction of ROS, and upregulation of HO-1 protein after fasudil treatment.

Netrins are secreted molecules involved in axon guidance and angiogenesis. However, the role of netrins in the vasculature remains unclear. Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors. Previously, we found that netrin-1 acts as an anti-angiogenic factor in rats by inhibiting alkali burn-induced corneal neovascularization. Here, we further investigate the effects of netrin-4, another member of the same netrin family, on neovascularization in vitro and in vivo. We found that netrin-4 functions similarly as netrin-1 in angiogenesis. In vitro angiogenesis assay shows that netrin-4 affected human umbilical vein endothelial cell (HUVEC) tube formation, viability and proliferation, apoptosis, migration, and invasion in a dose-dependent manner. Netrin-4 was topically applied in vivo to alkali-burned rat corneas on day 0 (immediately after injury) and/or day 10 post-injury. Netrin-4 subsequently suppressed and reversed corneal neovascularization. Netrin-4 inhibited corneal epithelial and stromal cell apoptosis, inhibited vascular endothelial growth factor (VEGF), but promoted pigment epithelium-derived factor (PEDF) expression, decreased NK-KB p65 expression, and inhibits neutrophil and macrophage infiltration. These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues.

Background/aims: To report the influence of histological features of corneal surface pannus following ocular surface burn on the outcome of cultivated limbal epithelial transplantation (CLET)., Methods: On retrospectively reviewing the medical records of the patients who underwent autologous CLET from April 2002 to June 2012 at L V Prasad Eye Institute, Hyderabad, India, we could trace the histological reports in only 90 records. These 90 records, besides clinical parameters, were reviewed for the influence of various histological features on the final outcome of CLET., Results: The histological features include epithelial hyperplasia (21.1%), surface ulceration (2.2%), goblet cells (62.2%), squamous metaplasia (11.1%), active fibrosis (31.1%), severe inflammation (8.9%), multinucleated giant cells (3.3%), stromal calcification (8.9%) and active proliferating vessels (5.6%). Among these histological features, patients with either hyperplasia or calcification in their excised corneal pannus show an unfavourable outcome compared with patients without hyperplasia (p=0.003) or calcification (p=0.018). A similar unfavourable outcome was not seen with other histological features and various clinical parameters., Conclusions: Presence of either calcific deposits or hyperplasia in the excised corneal pannus provides poor prognostication; hence, a proper counselling of such patients is mandatory along with a close follow-up., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: Platelet-activating factor (PAF) has been found in various ocular tissues; the activity of PAF depends on the binding to its specific receptor, PAF-receptor. We investigated the therapeutic effects of PAF-receptor antagonists (CV-3988 and Ginkgolide B) on alkali burn-induced corneal neovascularization (CNV)., Methods: CNV was induced by applying a 0.2 N sodium hydroxide (3 µl, NaOH) solution directly on mice corneas. CV-3988 (1 mM/10 µl) and Ginkgolide B (1 mM/10 µl) were administered topically on the corneas three times daily for three consecutive days. CNV was evaluated under a slit-lamp microscope. Corneas were processed for histological, immunohistochemical and reverse transcription polymerase chain reaction analysis. Human umbilical vein endothelial cells were used for the migration and tube formation assay., Results: Application of CV-3988 and Ginkgolide B inhibited CNV caused by alkali burn. CV-3988 and Ginkgolide B attenuated the expression of PAF-receptor mRNA. Alkali injury induced a massively increased intraocular mRNA expression of an angiogenic factor in cornea tissues, whereas these increments were attenuated by the application of CV-3988 and Ginkgolide B., Conclusions: CV-3988 and Ginkgolide B reversed opacity and neovascularization in alkali burn-induced corneas. Our findings suggest that CV-3988 and Ginkgolide B may be therapeutically useful in the treatment of CNV and inflammation.

Background: To study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis., Methods: An alkali injury was produced with a filter paper of 3 mm with 1 N NaOH during 40 seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n = 12), Group 2, with 10 ul of SLPI (200 ug/ml; n = 12) and Group 3 was treated with buffer (10 ul; n = 12) topically administered four times a day for up to 7 days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24 hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence., Results: Corneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18 hours and 24 hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7 days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC., Conclusions: The binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.

Purpose: Recently, RAP1 (Telomeric Repeat Binding Factor 2, Interacting Protein [TERF2IP]) was discovered as a modulator that selectively regulates nuclear factor light chain kappa enhancer of activated B cells (NFκB) signaling. The roles of RAP1 in regulation of inflammation and angiogenesis for corneal recovery following corneal injury remain poorly understood. The effects of RAP1 deletion on corneal resurfacing and neovascularization in a corneal alkali burn mouse model were examined., Methods: Corneal defects and neovascularization were induced in vivo by infliction of an alkali burn to the cornea with 1 N sodium hydroxide solution in RAP1 knock-out (RKO) and wild-type (RWT) mice. Corneal resurfacing was evaluated using slit-lamp biomicroscopy. Neovascularization following injury was evaluated by bright view biomicroscopy and immunofluorescence staining with the endothelial marker platelet endothelial cell adhesion molecule (PECAM). The cytokine profiles of corneal tissue involved in inflammation and neovascularization following injury was compared between RKO and RWT mice. Corneal epithelial cells were isolated for classic scratch wound healing assay and further testing with lipopolysaccharide stimulation., Results: Resurfacing of the burned cornea was accelerated and angiogenesis was suppressed, faster recovery of corneal epithelial cells from classic scratch wound healing and superior tolerance of lipopolysaccharides challenge was observed in the RKO compared to RWT. Molecular investigation revealed that deletion of RAP1 reduced upregulation of inflammatory cytokine (IL1A), finely regulated the expression of angiogenic factor (VEGF), and antiangiogenic factor (PEDF), following injury for better corneal recovery., Conclusions: Deficiency of RAP1 facilitates corneal recovery after injury. Specificity of RAP1 inhibition may lead to design of specific inhibitors of NFκB in the treatment of ocular injuries., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Background: To evaluate the effects of sub-conjunctivally applied interleukin-6 receptor (IL-6R) antibody (tocilizumab) on alkali burn induced corneal neovascularization (CNV) in rats., Methods: Alkali burn induced corneal neovascularization was created in 24 right eyes of 24 rats. The rats were then randomized into 2 groups. Group 1 received sub-conjunctival injection of 4 mg/0.2 ml tocilizumab and Group 2 received sub-conjunctival injection of 0.2 ml normal saline at the 5th day of alkali burn. The corneal surface area invaded with neovascular vessels were calculated on photographs. The rats were sacrificed and the corneas were excised at the15th day. The corneal specimens were stained with hemotoxylin-eosin to evaluate tissue morphology and with Willebrand factor (vWF) to evaluate microvascular structures immunohistochemically. Vascular endothelial growth factor (VEGF) expression was analyzed by ELISA., Results: The percent area of CNV was 26.9% in Group 1 and 56.5% in Group 2 (p < 0.001). The histological evaluation showed that the corneal structures were not visibly altered by sub-conjuntival tocilizumab injection. Group 1 showed significantly lower corneal inflammation score than Group 2 (p < 0.001). The number of vessels stained with vWF were significantly higher in Group 2 than Group 1 (15.23 and 5.46, respectively; p < 0.001). ELISA analyses showed that corneal VEGF levels were significantly lower in Group 1 compared to Group 2 (p = 0.013) CONCLUSION: The present data demonstrated first time the beneficial effects of sub-conjunctival tocilizumab on decreasing CNV in alkali burn model of the rat cornea. Further studies are warranted to confirm these findings for the clinical application.

Purpose: To evaluate the therapeutic effect of human adipose-derived stem cells (hASCs) overlaid on a scleral contact lens (SCL) carrier in a rabbit model of ocular alkaline burn., Materials and Methods: After inducing alkaline burn in 11 New Zealand white rabbits, hASCs cultured on SCLs were placed on the right eye of 5 rabbits, SCLs without cells were used in 5, and no treatment was applied in 1 eye. Each eye was examined and photographed for corneal vascularization, opacities, and epithelial defect in week 1, 2, and 4 after surgery. After 1 month, rabbits were killed and the corneas were removed and cut in half for electron and light microscopy examination., Results: Human adipose-derived stem cells were attached to SCL surface and confluent easily. Human adipose-derived stem cells on SCL eyes showed smaller epithelial defect, less corneal opacity, corneal neovascularization relative to SCL eyes. Both groups showed no symblepharon. However, the cornea in the untreated eye was melted in 2 weeks and developed severe symblepharon., Conclusion: Human adipose-derived stem cells on SCL can reduce inflammation and corneal haziness in severe ocular alkaline burn injury in rabbits.

The purpose of the present study was to quantify and compare corneal hem- and lymphangiogenesis between alkali burn and suture-induced corneal neovascularization (CNV) in two commonly used mouse strains. A retrospective analysis was performed on C57BL/6 and FVB neovascularized corneas. CNV was induced by surface caustication with NaOH or intrastromal placement of three 10.0 nylon sutures. Hemangiogenesis and lymphangiogenesis extent was calculated on whole mounted corneas by CD31 and LYVE1 immunofluorescence analysis. Blood vessel growth was similar between alkali burn and suture-induced CNV in C57BL/6 mice, and between C57BL/6 and FVB sutured strains. On the contrary, corneal lymphangiogenesis was more pronounced in the C57BL/6 sutured mice versus the alkali burn group, and in the FVB strain versus both C57BL/6 models. These results indicate that significant differences occur in lymphangiogenesis, but not hemangiogenesis, in the alkali burn and suture-induced models in C57BL/6 mice. Furthermore, lymphangiogenesis is more pronounced in the albino (FVB) strain after suture placement. We suggest that the suture model has a number of advantages and may be preferentially used to study corneal lymphangiogenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: To observe the effect of TC14012 (a CXCR4 antagonist and CXCR7 agonist) on alkali burn-induced corneal neovascularization (CNV) in a mouse model., Methods: CNV was induced in vivo by alkali burns on the corneas of BALB/c mice. A total of 54 mice treated with alkali burns were randomly divided into 3 groups, each of which received one of the following treatments: bilateral subconjunctival injections of TC14012 for 3 consecutive days, bilateral subconjunctival injections of balanced saline (BS) for 3 consecutive days or no treatment (blank control). The areas of CNV were measured on days 3, 7 and 14 after the alkali burns. CXCR4, CXCR7, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) mRNAs were detected and quantified by real-time reverse transcription PCR on days 7 and 14. Additionally, the expression of the proteins CXCR4, CXCR7, VEGF, β-arrestin 2, total ERK1/2 and phospho-ERK1/2 was determined by Western blotting., Results: On day 7 after the alkali burns, the CNV area, VEGF, MMP-2 and MMP-9 mRNA levels, and VEGF, β-arrestin 2 and phospho-ERK1/2 protein levels were increased in the TC14012 group compared with the nontreatment and BS groups. However, on day 14, the CNV area, CXCR4, CXCR7, VEGF, MMP-2 and MMP-9 mRNA levels, and the CXCR4, CXCR7, VEGF and β-arrestin 2 protein levels were significantly decreased in the TC14012 group., Conclusions: TC14012 initially enhanced alkali burn-induced CNV but reduced CNV in later stages. In addition to CXCR4, CXCR7 is involved in the pathogenesis of CNV., (© 2014 S. Karger AG, Basel.)

Purpose: We clarified the effects of an ophthalmic solution of a peroxisome proliferator-activated receptor gamma (PPARγ) agonist on corneal inflammation and wound healing after alkali burn injury in rats., Methods: After alkali exposure, either an ophthalmic solution with 0.1% pioglitazone hydrochloride (the PPARγ group) or vehicle (the vehicle group) was topically applied to the cornea until day 14. Histological, immunohistochemical, and real-time reverse transcription polymerase chain reaction analysis were performed., Results: After alkali injury, PPARγ expression increased, with the infiltration of many inflammatory cells. The infiltration of neutrophils and macrophages started from the corneal limbus within 6 h, and developed in the corneal center by day 7, with associated neovascularization. The accumulation of α-smooth muscle actin-positive myofibroblasts and the deposition of type III collagen were noted on day 14. The histological changes were suppressed significantly by treatment with the ophthalmic solution of the PPARγ agonist. In addition, the number of infiltrating M2 macrophages in the cornea was increased by PPARγ agonist treatment. In real-time reverse transcription polymerase chain reaction analysis, the messenger ribonucleic acid expression levels of interleukin-1β (IL-1β), IL-6, IL-8, monocyte chemoattractant protein-1, tumor necrosis factor-α, transforming growth factor beta 1, and vascular endothelial growth factor-A were decreased in the PPARγ group compared to the vehicle group in the early periods of corneal inflammation., Conclusions: The ophthalmic solution of the PPARγ agonist inhibited inflammation, decreased the fibrotic reaction, and prevented neovascularization in the cornea from the early phase after alkali burn injury. The ophthalmic solution of the PPARγ agonist may provide a new treatment strategy with useful clinical applications for corneal inflammation and wound healing.

Purpose: To explore the inhibitory effects of doxycycline on allograft rejection in alkali-burned cornea beds., Methods: The corneas of BALB/c mice were injured using a 1 mol/l NaOH solution. Following the injury, the corneas from C57BL/6 mice were transplanted into the eyes of BALB/c mice after being randomized into three groups: allogeneic corneal transplantation (group A), topical use of doxycycline after allogeneic corneal transplantation (group B) and syngeneic corneal transplantation (group C). Corneal angiogenesis was examined using whole-mount immunofluorescence, and corneal inflammation was evaluated using inflammation index scoring. The immune rejection of the grafts was examined using a slit lamp. In addition, the expression of vascular endothelial growth factor A and interleukin-1β in the transplanted corneas was examined using a real-time polymerase chain reaction, immunohistochemistry and an enzyme-linked immunosorbent assay., Results: The outgrowth of the corneal blood vessels in the group A mice was faster than that in the group B and group C mice. The inflammation index levels were highest in the group A mice, intermediate in the group B mice and lowest in the group C mice. Vascular endothelial growth factor and the interleukin-1β protein and mRNA levels decreased dramatically in the group B mice compared with the group A mice (all p-values < 0.01). In addition, the mean survival time in the group B mice (27.00 ± 2.00 days) was significantly longer than that in the group A mice (11.67 ± 1.51 days; p < 0.05)., Conclusions: Doxycycline may have had a significant role in preventing corneal angiogenesis and inflammation in alkali-burned corneal beds, which resulted in higher allograft survival rates., (© 2013 The Authors. Acta Ophthalmologica © 2013 Acta Ophthalmologica Scandinavica Foundation. Published by Blackwell Publishing Ltd.)

Epidermal growth factor (EGF) is used to treat alkali-burned corneas. However, EGF-induced corneal angiogenesis, which is currently untreatable, is a side effect of this therapy. We therefore explored the role of the intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) in EGF-induced angiogenesis and tested whether KCa3.1 blockade can suppress EGF-induced corneal angiogenesis. The proliferation, migration and tube formation of HUVECs (human umbilical vein endothelial cells) in response to EGF, the MEK inhibitor PD98059 and the KCa3.1 inhibitor TRAM-34 were analyzed in vitro via MTT, cell counting, scratch and tube formation assays. The protein and mRNA levels of KCa3.1, phosphorylated-ERK (P-ERK), total-ERK (T-ERK), cyclin-dependent kinase 4 (CDK4), vimentin and MMP-2 were assessed via western blotting and RT-PCR. KCa3.1 and vimentin expression were also detected through immunofluorescence staining. Flow cytometry was performed to examine the cell cycle. Further, an in vivo murine alkali-burned cornea model was developed and treated with EGF and TRAM-34 eye drops to analyze the effect of these treatments on corneal healing and angiogenesis. The corneas were also analyzed by histological staining. The in vitro results showed that EGF induces the upregulation of KCa3.1 and P-ERK in HUVECs and that this upregulation is suppressed by PD98059. EGF stimulates proliferation, migration and tube formation in HUVECs, and this effect can be suppressed by TRAM-34. TRAM-34 also arrests HUVECs in the G1 phase of the cell cycle and downregulates CDK4, vimentin and MMP-2 in these cells. The in vivo results indicated that TRAM-34 suppresses EGF-induced corneal angiogenesis without affecting EGF-induced corneal wound healing. In summary, the upregulation of KCa3.1 may be crucial for EGF-induced angiogenesis through the MAPK/ERK signaling pathway. Thus, KCa3.1 may be a potential target for the treatment of EGF-induced corneal angiogenesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Ocular exposure to mustard agents may cause severe and prolonged injury to the anterior segment tissues. Effective decontamination of the external eye surface after exposure is of paramount importance. The purpose of the present study was to assess the effectiveness of Diphoterine rinsing solution (DRS) in reducing ocular damage after exposure to nitrogen mustard (NM) and to compare it with normal saline solution., Methods: One eye of 16 New Zealand albino rabbits was exposed to 2% NM. Immediate thorough irrigation was performed with either 500 ml of DRS (treated group) or with 500 ml of normal saline (control group). The magnitude of ocular injury and response to treatment were assessed by examiners masked to the treatment assignment during 22 days following the exposure., Results: Immediate ocular irrigation with DRS was more effective compared with saline in reducing corneal, iris and anterior chamber injury. In the DRS-treated group, the corneal opacity and corneal neovascularization were less severe, and development of iris atrophy was delayed. Intraocular pressure (mmHg) was better maintained when compared to the control group (day 7 24.3 versus 14.8, p = 0.003; day 12 28 versus 15, p = 0.003; day 22 33.5 versus 21.8, p = 0.014, respectively). Systemic oxidative stress associated with exposure to NM was significantly higher in the saline-treated group than in DRS-treated group (p < 0.011)., Conclusions: The findings of this study indicate the effectiveness of DRS in reducing of NM-induced ocular injuries. Its use should be considered as an immediate treatment modality following exposure to mustard agents to reduce potential ocular injury., (© 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.)

Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis, and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways, and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders.

Purpose: We analyzed the expression of angiogenesis-related factors in corneal tissues that had undergone previously autologous cultivated oral mucosal epithelial transplantation (COMET)., Methods: Six eyes from four chemically- and two thermally-injured patients with limbal stem cell deficiency who received COMET to promote wound healing were studied retrospectively. Immunoconfocal microscopy was performed on corneal specimens from the patients after COMET, as well on normal corneas, conjunctiva, and oral mucosa for keratin 8, fibroblast growth factor-2 (FGF-2), VEGF, collagen XVIII (endostatin), pigment epithelium-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), tissue inhibitor of metalloproteinase-3 (TIMP-3), thrombospondin-1 (TSP-1), and interleukin-1 receptor antagonist (IL-1ra)., Results: FGF-2, VEGF, endostatin, PEDF, and IL-1ra were detected in all the samples, with signals for FGF-2, VEGF, and IL-1ra localized to the full-thickness epithelial layer, as signals for endostatin limited to the basement membrane. Expression of PEDF varied in tissues, with a preferential expression in the suprabasal epithelial layer. FGF-2 and IL-1ra were abundantly expressed in the basal epithelial layer in specimens with increased stratification. Signals for sFlt-1, TIMP-3, and TSP-1 were detected in normal corneal epithelium, and in a specimen containing corneal epithelium, but were negative in all other specimens., Conclusions: Expression of FGF-2, VEGF, PEDF, endostatin, and IL-1ra was similar in normal corneas, conjunctiva, oral mucosa, and corneas after COMET. Expression of sFlt-1, TIMP-3, and TSP-1 was limited to normal corneas and negative for other tissues. A lack of the aforementioned antiangiogenic factors may contribute to the peripheral corneal neovascularization seen after COMET.

Cellular senescence acts as a potent regulator of tumor suppression and fibrosis limitation; however, its contribution and crosstalk with neovascularization during normal wound healing has not been examined. Here, we explored the role of senescent fibroblasts on neovascularization with a mouse model of alkali-induced corneal wound healing. Senescent cells accumulated in corneal stroma from day 7 to 27 after alkali burn and peaked on day 14, which was consistent with the development of corneal neovascularization (CNV). In vitro and in vivo assays confirmed that the senescent cells were derived primarily from activated corneal fibroblasts. Furthermore, senescent corneal fibroblasts exhibited enhanced synthesis and secretion of extracellular matrix-degrading enzymes (matrix metalloproteinases 2, 3, and 14 and tissue- and urokinase-type plasminogen activators) and angiogenic factors (vascular endothelial growth factor) and decreased expression of anti-angiogenic factors (pigment epithelium-derived factor and thrombospondins), which supported the proliferation, migration, and promotion of tube formation of vascular endothelial cells. Intrastromal injection of premature senescent fibroblasts induced CNV earlier than that of normal fibroblasts, while matrix metalloproteinase inhibitors blocked the early onset of senescent cell-induced CNV. Therefore, senescent fibroblasts promoted the alkali-induced CNV partially via the enhanced secretion of matrix metalloproteases., (Copyright © 2011 Wiley Periodicals, Inc.)

Purpose: To evaluate the efficacy and safety of subconjunctival injection of triamcinolone in the treatment of acute ocular alkali burn in rabbits., Methods: Two groups of 5 rabbits were subjected to alkali burn (1 N NaOH). One group was treated with 1 subconjunctival injection of 0.3 mL of triamcinolone and the other with 1 subconjunctival injection of 0.3 mL of 0.9% saline. The affected corneas were observed for vascularization and opacity approximately 10 minutes after the burn and also after 7, 14, and 21 days. Photographs were taken for observation and statistical analyses. At all time intervals, the corneas were classified according to predetermined scores. Twenty-one days after the treatment, the animals were anesthetized, and their eyes were enucleated and processed for histopathology., Results: Greater vascularization and opacity appeared in the animals that were treated with saline than in those treated with subconjunctival triamcinolone (vascularization: 7 days, P = 0.0107; 14 days, P = 0.0099; and 21 days, P = 0.0088; opacity: 7 days, P = 0.0079; 14 days, P = 0.0112; and 21 days, P = 0.0255). These results were also compatible with the morphological and statistical analyses, which revealed a more intense inflammatory process in the group treated with saline (P = 0.0317). No complications, such as corneal melting, perforation, or infection, were observed., Conclusions: Subconjunctival injection of triamcinolone may be a therapeutic option for the treatment of acute ocular burn because it reduced the corneal inflammatory process, opacity, and vascularization, with no apparent clinical changes in the general state of the animal.

Purpose: To investigate the role of reactive oxygen species (ROS) as the prime initiators of the angiogenic response after alkali injury of the cornea and observe the effects of antioxidants in preventing angiogenesis., Methods: The corneal epithelia of SOD-1-deficient mice or wild-type (WT) mice were removed after application of 0.15 N NaOH to establish the animal model of alkali burn. ROS production was semiquantitatively measured by dihydroethidium (DHE) fluorescence. Angiogenesis was visualized by CD31 immunohistochemistry. The effects of the specific NF-κB inhibitor DHMEQ, the antioxidant N-acetyl-L-cysteine (NAC), and hydrogen (H2) solution were observed., Results: ROS production in the cornea was enhanced immediately after alkali injury, as shown by increased DHE fluorescence (P<0.01). NF-κB activation and the upregulation of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were significantly enhanced (P<0.01), leading to a significantly larger area of angiogenesis. Angiogenesis in SOD-1-/- mice corneas were significantly higher in WT mice (P<0.01), confirming the role of ROS. Pretreatment with the specific NF-κB inhibitor DHMEQ or the antioxidant NAC significantly reduced corneal angiogenesis by downregulating the NF-κB pathway (P<0.01) in both WT and SOD-1-/- mice. Furthermore, we showed that irrigation of the cornea with hydrogen (H2) solution significantly reduced angiogenesis after alkali-burn injury (P<0.01)., Conclusions: Immediate antioxidant therapy with H2-enriched irrigation solution is a new potent treatment of angiogenesis in cornea to prevent blindness caused by alkali burn.

Objective: To examine the effect of recombinant canstatin protein on the corneal neovascularization (CorNV) in an alkaline burn-induced CorNV model., Methods: This study involved 50 C57BL/6 mice. CorNV was induced by an alkaline burn of the corneas with 1 N NaOH under general anesthesia. Beginning 24 h after CorNV induction, recombinant canstatin protein was administered intraperitoneally at 5 or 10 mg/kg body weight once a day for up to 14 days. CorNV was evaluated by slit-lamp microscopy. Growth factors and cytokines relating to neovascularization and inflammation in the corneas were evaluated by real-time polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry or ELISA., Results: Recombinant canstatin protein significantly inhibited CorNV. Compared to the untreated or PBS-treated CorNV group, expression of vascular endothelial growth factor (VEGF) markedly decreased in the canstatin-treated group as detected by various methods. Western blotting and RT-PCR showed that the canstatin treatment inhibited the expression of hypoxia-inducible factor and VEGF. Day 7 revealed the greatest changes: ELISA assay showed that TNF-α also significantly decreased in canstatin-treated corneas., Conclusions: Recombinant canstatin protein suppressed experimental CorNV, suggesting that canstatin may serve as a useful angiogenic inhibitor for the treatment of neovascularization-related corneal diseases., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: Suture placement and alkali burn to the cornea are often used to induce inflammatory corneal neovascularization (CorNV) models in animals. This study compares the changes in genome-wide gene expression under these two CorNV conditions in mice., Methods: CorNV were induced in Balb/c mice by three interrupted 10-0 sutures placed at sites about 1 mm from the corneal apex, or by alkali burns that were 2 mm in size in the central area of the cornea. At the points in time when neovascularization progressed most quickly, some eyeballs were subjected to histological staining to examine CorNV and inflammatory cells infiltration, and some corneas were harvested to extract mRNA for microarray assay. After normalization and filtering, the microarray data were subject to statistical analysis using Significance Analysis of Microarray software, and interested genes were annotated using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) program. The expression change of classical proangiogenic molecule like vascular endothelial growth factor (VEGF) and antiangiogenic molecule like pigment epithelium-derived factor (PEDF) was further verified using western blotting., Results: Suture placement induced CorNV in the areas between the suture and limbus, but did not affect the transparency of the yet unvasuclarized areas of the corneas. In contrast, alkali burn caused edema and total loss of transparency of the whole cornea. Histology showed that sutures only caused localized epithelial loss and inflammatory infiltration between the suture and limbus, but chemical burn depleted the whole epithelial layer of the central cornea and caused heavy cellular infiltration of the whole cornea. At day 5 after suture placement, 1,055 differentially expressed probes were identified, out of which 586 probes were upregulated and 469 probes were downregulated. At a comparable time point, namely on day 6 after the alkali burn to the corneas, 472 probes were upregulated and 389 probes were downregulated. Among these differentially expressed probes, a significant portion (530 probes in total, including 286 upregulated and 244 downregulated probes) showed a similar pattern of change in both models. Annotation (using DAVID) of the overlapping differential genes revealed that the significant enrichment gene ontology terms were "chemotaxis" and "immune response" for the upregulated genes, and "oxidation reduction" and "programmed cell death" for the downregulated genes. Some genes or gene families (e.g., S100A family or α-, β-, or γ-crystallin family) that had not been related to corneal pathogenesis or neovascularization were also revealed to be involved in CorNV. VEGF was upregulated and PEDF was stable as shown with western blotting., Conclusions: Sutures and alkali burn to the corneas produced types of damage that affected transparency differentially, but gene profiling revealed similar patterns of changes in gene expression in these two CorNV models. Further studies of the primary genes found to be involved in CorNV will supplement current understanding about the pathogenesis of neovascularization diseases.

Purpose: To investigate the effect of bevacizumab in a model of corneal inflammation., Materials and Methods: Epithelium from the cornea and limbus was completely removed using 100% alcohol in rats. Bevacizumab (1.25 mg/0.05 ml) or normal saline was subconjunctivally injected. One week later, corneas were examined and subjected to hematoxylin-eosin and immunofluorescent staining for VEGF. Expression of IL-2, IFN-gamma, IL-6, and TGF-beta 1 were analyzed by ELISA., Results: Bevacizumab showed a borderline reduction in corneal neovascularization (11.0 +/- 4.8% and 18.0 +/- 10.0% in the bevacizumab and control groups, respectively; p = 0.054), while the extent of epithelialization was not affected (5.0 +/- 3.4% and 6.1 +/- 5.2% in the bevacizumab and control groups, respectively; p = 0.715). The infiltration of inflammatory cells was reduced (99.3 +/- 22.3 cells/x 400 in the bevacizumab-injected corneas and 182.3 +/- 58.0 cells/x 400 in the controls; p = 0.013). The levels of IL-2, IFN-gamma, and IL-6 were decreased in the rats with the bevacizumab injections (44 +/- 6 and 79 +/- 9 pg/ml for IL-2 in the bevacizumab-injected group and control, respectively, p = 0.025; 45 +/- 5 and 67 +/- 6 pg/ml for IFN-gamma in the bevacizumab-injected group and controls, respectively, p = 0.043; 45 +/- 6 and 75 +/- 8 pg/ml for IL-6 in the bevacizumab-injected group and controls, respectively, p = 0.030)., Conclusions: Bevacizumab showed a reduction in inflammatory cell infiltration and cytokines in chemically burned rat corneas.

Purpose: The four-and-one-half LIM domain-containing protein2 (FHL2) is a member of the four-and-a-half LIM domain-only (FHL) gene family. Although FHL2 is expressed in the cornea, its role in angiogenesis is unclear. The aim of this study was to investigate the role of the FHL2 gene in corneal angiogenesis after chemical injury., Methods: FHL2-LacZ knock-in mice were used to trace FHL2 gene expression before and after corneal injury. Corneal angiogenesis between FHL2-null mice and wild-type mice that underwent chemical and mechanical denudation of corneal and limbal epithelium were compared. New growth vessel density was assessed by CD31 staining and was analyzed using image analysis software. Levels of vascular endothelial growth factor (VEGF) and cyclooxygenase (COX)-2 proteins were determined by Western blot assay., Results: beta-Galactosidase staining of corneal tissue in FHL2-LacZ knock-in mice revealed that FHL2 gene expression is upregulated in the corneal epithelium after corneal injury. Ten days after injury, corneal neovascularization was observed in control and FHL2-null mice. New corneal vessel density was found to be lower in the FHL2-null mice injury group than in the wild-type mice injury group. Western blot analysis showed that VEGF and COX-2 protein levels were higher after chemical injury in FHL2-null mice and wild-type mice. However, the upregulated VEGF protein was significantly lower in the FHL2-null mice than in the wild-type mice., Conclusions: The decreased chemical-induced corneal angiogenesis found in the FHL2-null mice in this study indicated that FHL2 protein plays a role in inhibiting inflammatory angiogenesis.

Purpose: To examine the efficacy of oral doxycycline treatment (as compared to oral and topical dexamethasone) for inhibiting corneal neovascularization (CNV)., Methods: Following corneal alkali burn, rats were treated daily with oral doxycycline, oral dexamethasone, or topical dexamethasone for 14 days. Control rats were injured but were not treated. At days 3, 7, and 14 post injury, length and area of CNV were evaluated, as well as corneal epithelial healing and ulceration. Tissues were obtained from a subset of rats from each group for histopathological analysis., Results: At days 7 and 14 post-injury, CNV length in the doxycycline group was significantly less than in the untreated control (p < 0.008). The area of CNV was significantly smaller in doxycycline as compared to control rats on days 3, 7, and 14 post-injury. Inhibition of CNV (indicated by area and length) was significantly greater in both dexamethasone groups compared to the doxycycline group (p < 0.008 for all comparisons). However, epithelial healing was significantly more rapid in the doxycycline group compared to both dexamethasone groups (p < 0.008). Epithelial ulceration was apparent in both oral and topically treated dexamethasone rats, but not in doxycycline-treated rats., Conclusions: Oral doxycycline inhibits CNV without the harmful side effects associated with dexamethasone use. Further investigation is warranted to assess the mechanisms through which doxycycline acts to cause CNV inhibition, and the applicability of doxycycline use for treating CNV in the clinical setting.

Purpose: To determine whether long-term expression of intraceptors can be achieved using plasmid albumin nanoparticles and whether nanoparticles can inhibit and cause regression of murine corneal neovascularization induced by mechanical-chemical trauma., Methods: Albumin nanoparticles encapsulating pCMV.Flt23K were developed as a lyophilized product that is easily redispersed in an aqueous medium. Nanoparticles were injected into the corneas of uninjured BALB/c mice and observed for toxicity for 3 weeks. Entry of nanoparticles into corneal cells was demonstrated through transmission electron microscopy and confocal imaging. Naked pCMV.Flt23K, nanoparticles encapsulating pCMV.Flt23K, or empty pCMV nanoparticles were injected into uninjured mouse corneas. These corneas were subjected to mechanical alkali trauma 3 weeks after injection., Results: Nanoparticles were nontoxic to the cornea and entered into corneal keratocyte cytoplasm. They persisted for at least 4 weeks in the cornea, expressed effective intraceptor levels for at least 5 weeks, and reduced corneal neovascularization by approximately 40% (P = 0.035) at 5 weeks after administration., Conclusions: Albumin nanoparticles are not toxic to the cornea and can express intraceptors for extended periods that are effective in suppressing injury-induced corneal neovascularization.

Purpose: Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. The purpose of the present study was to evaluate the therapeutic effect of K5 on alkali-burn-induced corneal neovascularization (NV) and to investigate its mechanism of action., Methods: Corneal NV was induced in rabbits by NaOH. The rabbits received eye drops containing K5 or vehicle alone, four times per day. Corneal NV and inflammation were monitored every other day with a slit lamp microscope, and the length of the vessels in the cornea and the area of NV were measured. Vascular endothelial growth factor (VEGF) was determined by immunohistochemical and Western blot analyses. The TUNEL assay was used to assess the apoptosis of endothelial cells. The effects of K5 on primary bovine aortic endothelial cells (BAECs) were determined by MTT assay, flow cytometry, transmission electron microscopy, and DNA fragmentation assay., Results: Alkali-burn-induced progressive corneal NV and inflammation in the cornea. K5 delayed the onset of corneal NV (P < 0.05) and decreased NV areas (P < 0.05) in a dose-dependent manner. K5 treatment, after the formation of corneal NV, induced regression of newly formatted vessels in the cornea. K5 decreased the inflammatory index in the corneas at different time points after the alkali burn. Corneal VEGF levels were reduced by K5 treatment. K5 inhibits proliferation and induces apoptosis in BAECs., Conclusions: Topical application of K5 may have therapeutic potential for the chemical burn-induced corneal NV and inflammation. The inhibitory effect of K5 on corneal NV may be by downregulation of VEGF expression.

The present paper deals with the study of the efficiency of oral use of the antioxidative drug Immugen (a complex of alpha-tocopherol, oubichinone, selenium aspartate, methionine, and soyabean phospholipids) on a rabbit model of severe alkaline-induced corneal burn. The investigations have indicated that addition of Immugen to the rabbit feed exerts a significant positive effect on the parameters of the local antioxidative system of the eye and causes an increase in the activity of superoxide dismutase and catalase, and, on day 14, in antioxidative activity. The early experimental periods were marked by a slight rise in the frequency of deep corneal ulcerations. Moreover, the long-term clinical effect of use of Immugen appears as a significant increase in the area of the transparency-preserving affected cornea. The findings suggest that the antioxidants can show their optimal effect in the complex therapy for burn processes, including the use of proteinase inhibitors.