The effect of lactoferin on the free radical and cytokine status of cornea in the experimental thermal burn.

The free radical and cytokine statuses of the cornea during its thermal burn and the possibility of its correction by lactoferrin have been studied in Soviet Chinchilla rabbits. The development of a corneal thermal burn was accompanied by the development of oxidative stress (increased levels of TBA-reactive substances and carbonyl derivatives of proteins, decreased activity of SOD and GPx enzymes) and a pronounced inflammatory reaction with increased levels of TNF-1α, IL-10, TGF-1ß. The use of lactoferrin had a pronounced therapeutic effect, which was manifested by accelerated healing, prevention of the development of complications (corneal perforations), a decrease in the severity of oxidative stress, an increase in the concentrations of TNF-1α (in the early stages), IL-10 (in the later stages), TGF-1ß (throughout the experiment). At the same time, by the end of regeneration more severe corneal opacification was recognized compared to the control group. This may be associated with an increased level of anti-inflammatory cytokines, especially TGF-1ß.

Endogenous TSG-6 modulates corneal inflammation following chemical injury.

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

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.

An electrospun scaffold functionalized with a ROS-scavenging hydrogel stimulates ocular wound healing.

Ocular alkali burn is a serious ophthalmic emergency. Highly penetrative alkalis cause strong inflammatory responses leading to persistent epithelial defects, acute corneal perforation and severe scarring, and thereby persistent pain, loss of vision and cicatricial sequelae. Early and effective anti-inflammation management is vital in reducing the severity of injury. In this study, a double network biomaterial was prepared by compounding electrospinning nanofibres of thioketal-containing polyurethane (PUTK) with a reactive oxygen species (ROS)-scavenging hydrogel (RH) fabricated by crosslinking poly(poly(ethylene glycol) methyl ether methacrylate-co-glycidyl methacrylate) with thioketal diamine and 3,3'-dithiobis(propionohydrazide). The developed PUTK/RH patch exhibited good transparency, high tensile strength and increased hydrophilicity. Most importantly, it demonstrated strong antioxidant activity against H2O2 and 2,2-di(4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH). Next, a rat corneal alkali burn model was established, and the PUTK/RH patch was transplanted on the injured cornea. Reduced inflammatory cell infiltration was revealed by confocal microscopy, and lower expression levels of genes relative to inflammation, vascularization and scarring were identified by qRT-PCR and western blot. Fluorescein sodium dyeing, hematoxylin and eosin (H&E) staining and immunohistochemical staining confirmed that the PUTK/RH patch could accelerate corneal wound healing by inhibiting inflammation, promoting epithelial regeneration and decreasing scar formation. STATEMENT OF SIGNIFICANCE: Ocular alkali burn is a serious ophthalmic emergency, characterized with persistent inflammation and irreversible vision loss. Oxidative stress is the main pathological process at the acute inflammatory stage, during which combined use of glucocorticoids and amniotic membrane transplantation is the most widely accepted treatment. In this study, we fabricated a polyurethane electrospun nanofiber membrane functionalized with a ROS-scavenging hydrogel. This composite patch could be a promising amniotic membrane substitute, possessing with a transparent appearance, elasticity and anti-inflammation effect. It could be easily transplanted onto the alkali-burned corneas, resulting in a significant inhibition of stromal inflammation and accelerating the recovery of corneal transparency. The conception of ROS-scavenging wound patch may offer a new way for ocular alkali burn.

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.

Characterization of C-X-C chemokine receptor type 5 in the cornea and role in the inflammatory response after corneal injury.

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

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.

Suppression of the caspase-1/GSDMD-mediated pyroptotic signaling pathway through dexamethasone alleviates corneal alkali injuries.

The pathological mechanism of corneal injuries mediated by alkali burns are associated with Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 protein (NLRP3)-related corneal sterile inflammation. Whether the executive protein gasdermin D (GSDMD) of pyroptosis mediated by the NLRP3 inflammasome is present in alkali-induced corneal lesions remains unclear. Dexamethasone (Dex) is a commonly used drug for ocular surface diseases that can maintain corneal transparency and anti-inflammatory effects by topical administration. Here, we presented evidence that the effect of Dex on the pyroptosis-related caspase-1/GSDMD pathway in corneal alkali burns (CABs). We assessed the clinical manifestations and histological characteristics of the placebo group, 0.05% Dex group, 0.1% Dex group on day 3 or day 7 postburn and the control group (healthy corneas). The expression of factors (including NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N termini, pro-interleukin(IL)-1ß, IL-1ß, pro-IL-18 and IL-18) involved in the pyroptosis related caspase-1/GSDMD signaling pathway was demonstrated by molecular experiments in CAB. Alkali burns can upregulate the originally relatively dim expression of NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, pro-IL-1ß, pro-IL-18, IL-1ß and IL-18 in the healthy corneal epithelium and stroma. However, Dex can reverse the enhanced expression at the two timepoints. Corneal sterile inflammation can activate the NLRP3 inflammasome through the innate immune response mechanism and then activate the pyroptosis-related caspase-1/GSDMD signaling pathway. In addition, Dex can inhibit pyroptosis through this pathway.

Fibrin-Plasma Rich in Growth Factors Membrane for the Treatment of a Rabbit Alkali-Burn Lesion.

The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.

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.

Canonical NF-κB signaling maintains corneal epithelial integrity and prevents corneal aging via retinoic acid.

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; Relaf/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.

Extracellular Matrix Deposition and Remodeling after Corneal Alkali Burn in Mice.

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

Epigenetic Landscape Analysis of the Long Non-Coding RNA and Messenger RNA in a Mouse Model of Corneal Alkali Burns.

Purpose: Corneal alkali burns (CABs) are a common clinical ocular disease, presenting a poor prognosis. Although some long noncoding RNAs (lncRNAs) reportedly play a key role in epigenetic regulation associated with CABs, studies regarding the lncRNA signature in CABs remain rare and elusive. Methods: A CAB model was established in C57BL/6J mice and profiling of lncRNA expressions was performed by RNA-Seq. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related pathological pathways and candidate genes. RT-qPCR was used to verify the expression pattern of lncRNAs and related mRNAs, both in vitro and in vivo. Data were statistically analyzed by GraphPad Prism version 6.0. Results: In all, 4436 aberrantly expressed lncRNAs were identified in CAB mice when compared with control mice. In the top 13 aberrantly expressed lncRNAs, Bc037156 and 4930511E03Rik were confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway was most enriched. Following 4930511E03Rik siRNA treated, Srgn, IL-1ß and Cxcr2 were significant upregulated in corneal epithelial cells, corneal keratocytes, and bone marrow dendritic cells, with NaOH treatment. Moreover, after Bc037156 siRNA treated, expression levels of IL-1ß and Srgn were significantly downregulated in the three cell lines. Conclusions: Our study suggests that Bc037156 and 4930511E03Rik may be involved in inflammation, immune response, and neovascularization by regulating Srgn, IL-1ß, and Cxcr2 expression after CAB. These candidate lncRNAs and mRNAs may be the potential targets for the treatment strategy of the alkali injured cornea.

PLGA nanoparticles containing Lingzhi extracts rescue corneal epithelial cells from oxidative damage.

Oxidative stress-related ocular surface epithelial damage can be initiated by ambient oxygen, UV radiation, and chemical burns. The oxidative damage to cornea can lead to inflammation and even vision loss. Lingzhi (Ganoderma lucidum) is a Chinese herbal drug and has been shown to prevent chronic diseases in clinical practices and has been proven to possess anti-oxidative and anti-inflammatory properties. In the study, we prepared poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as a sustained drug release system of Lingzhi (LZH) to improve bioavailability. The particle size of developed NPs containing LZH (LZH-NPs) was ~184 nm with narrow size distribution. The results of cellular uptake revealed that using NPs as a drug delivery system could significantly increases the intracellular retention time. The results of the cell viability and chemiluminescence assay revealed that 5 µg/ml of LZH-NPs might be the threshold concentration for cultivation of corneal epithelial cells. After treating LZH-NPs in oxidative damaged cells, the results showed that the inflammation-related gene expression and DNA fragmentation level were both significantly decreased. Post-treatment of LZH-NPs in damaged corneal epithelial cells could increase the cell survival rate. In the rabbit corneal alkali burn model, topical instillation of LZH-NPs could promote corneal wound healing and decrease the inflammation. These results suggest that LZH-NPs may have the potential to treat ocular surface diseases caused by oxidative stress.

Ocular lamellar crystalline gels for sustained release and enhanced permeation of resveratrol against corneal neovascularization.

Corneal neovascularization (CNV) is the major cause of blindness after eye injury; however, only several drugs can be applied and the invasive administration ways (i.e., intravitreal injection and subconjunctival injection) are used. Resveratrol is a highly effective anti-VEGF agent against CNV. However, its applications are limited due to its strong hydrophobicity and instability. Here, we developed a resveratrol-loaded ocular lamellar crystalline gel (ROLG) for high inhibition of CNV. ROLGs were composed of resveratrol, glyceryl monooleate (GMO), ethanol, and water, and their lamellar crystalline structures were identified by polarizing light microscopy and small-angle X-ray scattering. High drug loading (4.4 mg/g) of ROLGs was achieved due to the hydrogen bonding between GMO and resveratrol. Resveratrol showed sustained release with 67% accumulative release in 7 h, which was attributed to the slow erosion of gels. Resveratrol in ROLGs had a high corneal permeation 3 times higher than resveratrol in hyaluronic acid suspensions (RHSs). ROLGs were administered to rats only once a day because of their strong retention on the cornea surface. ROLGs were safe due to the very little contact of ethanol in ROLGs to the cornea. CNV post-rat corneal alkaline injury was highly inhibited by ROLGs, resulting from the attenuation of corneal VEGF expression and then corneal healing was improved. The ROLG was a promising ocular medicine for the prevention of CNV.

Gelatin methacryloyl hydrogel eye pad loaded with amniotic extract prevents symblepharon in rabbit eyes.

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-ß1. 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-ß1 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.

S100A4 Silencing Facilitates Corneal Wound Healing After Alkali Burns by Promoting Autophagy via Blocking the PI3K/Akt/mTOR Signaling Pathway.

Purpose: This study investigated the role of S100 calcium binding protein A4 (S100A4) in corneal wound healing and the underlying mechanism of the S100A4-mediated PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. Methods: The rabbit corneal alkali burn model was established in vivo. S100A4 expression, wound healing, inflammation, and autophagy in rabbit cornea after alkali burn were detected. The NaOH-treated rabbit corneal stromal cells (rCSCs) were transfected with overexpressed S100A4 or silencing S100A4 to examine the effect of S100A4 on corneal wound healing in vitro. The effect of S100A4 on cell viability, proliferation, migration, invasion, fibrosis, and autophagy of rCSCs after alkali burn was analyzed. Then the functional rescue experiments were carried out. The PI3K inhibitor, LY294002, was used to elucidate the PI3K/Akt/mTOR signaling pathway in rCSCs. Results: S100A4 silencing promoted rabbit corneal wound healing by inhibiting fibrosis and inflammation and promoting autophagy in alkali-burned cornea, corresponding to increased levels of LC3, Beclin 1, and Atg4B but lowered α-smooth muscle actin, TNF-ɑ, and p62 levels. Moreover, silencing S100A4 inhibited proliferation, migration, invasion, and fibrosis of NaOH-treated rCSCs and promoted the differentiation of rCSCs into corneal cells and the autophagy of damaged rCSCs. The inhibitory role of S100A4 in wound healing was achieved via activation of the PI3K/Akt/mTOR pathway. Conclusions: S100A4 silencing confers a promising effect on wound healing of alkali-burned cornea by blocking the PI3K/Akt/mTOR pathway, supporting the advancement of corneal gene therapies for wound healing.

Combination of Peroxisome Proliferator-Activated Receptor (PPAR) Alpha and Gamma Agonists Prevents Corneal Inflammation and Neovascularization in a Rat Alkali Burn Model.

Peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) agonists have anti-inflammatory and anti-neovascularization effects, but few reports have tested the combination of PPARα and PPARγ agonists. In this study, we investigated the therapeutic effects of ophthalmic solutions of agonists of PPARα, PPARγ, and the combination in a rat corneal alkali burn model. After alkali injury, an ophthalmic solution of 0.05% fenofibrate (PPARα group), 0.1% pioglitazone (PPARγ group), 0.05% fenofibrate + 0.1% pioglitazone (PPARα+γ group), or vehicle (vehicle group) was topically instilled onto the rat's cornea twice a day. After instillation, upregulation was seen of PPAR mRNA corresponding to each agonist group. Administration of agonists for PPARα, PPARγ, and PPARα+γ suppressed inflammatory cells, neovascularization, and fibrotic changes. In addition, the PPARγ agonist upregulated M2 macrophages, which contributed to wound healing, whereas the PPARα agonist suppressed immature blood vessels in the early phase. Administration of PPARα+γ agonists showed therapeutic effects in corneal wound healing, combining the characteristics of both PPARα and PPARγ agonists. The results indicate that the combination of PPARα and γ agonists may be a new therapeutic strategy.

An Immunohistochemical Study of the Increase in Antioxidant Capacity of Corneal Epithelial Cells by Molecular Hydrogen, Leading to the Suppression of Alkali-Induced Oxidative Stress.

Corneal alkali burns are potentially blinding injuries. Alkali induces oxidative stress in corneas followed by excessive corneal inflammation, neovascularization, and untransparent scar formation. Molecular hydrogen (H2), a potent reactive oxygen species (ROS) scavenger, suppresses oxidative stress and enables corneal healing when applied on the corneal surface. The purpose of this study was to examine whether the H2 pretreatment of healthy corneas evokes a protective effect against corneal alkali-induced oxidative stress. Rabbit eyes were pretreated with a H2 solution or buffer solution, by drops onto the ocular surface, and the corneas were then burned with 0.25 M NaOH. The results obtained with immunohistochemistry and pachymetry showed that in the corneas of H2-pretreated eyes, slight oxidative stress appeared followed by an increased expression of antioxidant enzymes. When these corneas were postburned with alkali, the alkali-induced oxidative stress was suppressed. This was in contrast to postburned buffer-pretreated corneas, where the oxidative stress was strong. These corneas healed with scar formation and neovascularization, whereas corneas of H2-pretreated eyes healed with restoration of transparency in the majority of cases. Corneal neovascularization was strongly suppressed. Our results suggest that the corneal alkali-induced oxidative stress was reduced via the increased antioxidant capacity of corneal cells against reactive oxygen species (ROS). It is further suggested that the ability of H2 to induce the increase in antioxidant cell capacity is important for eye protection against various diseases or external influences associated with ROS production.

AMPK Activation by 5-Amino-4-Imidazole Carboxamide Riboside-1-ß-D-Ribofuranoside Attenuates Alkali Injury-Induced Corneal Fibrosis.

Purpose: Increased TGF-ß1 synthesis after corneal alkali injury is implicated in corneal fibrosis, as it promotes transdifferentiation of keratocytes into myofibroblasts. The activation of 5'-adenosine monophosphate-activated protein kinase (AMPK) by 5-amino-4-imidazole carboxamide riboside-1-ß-d-ribofuranoside (AICAR) inhibits TGF-ß1-induced fibrosis in other cell types. We investigated the antifibrotic effect of AICAR in corneal fibroblasts after alkali injury. Methods: Mouse models of corneal alkali burn, produced by placing 2-mm-diameter filter paper soaked in 0.1-N NaOH on the right cornea for 30 seconds, were treated with the test drugs 4× daily for 21 days. The central cornea was scanned by optical coherence tomography (OCT). Corneal tissues were obtained and processed for western blotting and immunohistochemistry. For in vitro analysis, primary human corneal fibroblasts were treated directly with TGF-ß1 to induce fibrosis, with or without AICAR pretreatment. Myofibroblast activation and extracellular matrix (ECM) protein synthesis were detected by western blotting, real-time PCR, and collagen gel contraction assay. Signaling proteins were analyzed by western blotting. Results: Alkali injury induced the upregulation of TGF-ß1 expression, which led to increased α-smooth muscle actin (α-SMA) and fibronectin synthesis and myofibroblast differentiation. AMPK activation by AICAR significantly suppressed TGF-ß1 and ECM protein expression. The antifibrotic effect of AICAR was AMPK dependent, as treatment with the AMPK inhibitor Compound C attenuated the antifibrotic response. Conclusions: AMPK activation by AICAR suppresses the myofibroblast differentiation and ECM synthesis that occur after alkali injury in corneal fibroblasts.