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

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

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.

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

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

Standardization of corneal alkali burn methodology in rabbits.

Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet's membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet's membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 µL of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet's membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.

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

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

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.

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

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

Effect of topical bovine colostrum in wound healing of corneal surface after acute ocular alkali burn in mice.

The purpose of this study was to evaluate the effect of bovine colostrum (BC) in the regeneration of corneal epithelial cells on an ocular alkali burn model. Twenty-four C57BL/6 mice were categorized into two gender/age-matched groups for treatment. Two days after inducing a corneal alkali burn in all left eyes with 4 µl of sodium hydroxide 0.15 mol/l, both eyes of group 1 were treated with BC 4 times per day, and both eyes of group 2 were treated with isotonic saline solution (SS). The epithelial defect was photographed and measured by fluorescein staining on days two, four, seven, and ten. Ocular burn damage was assessed with a pre-established classification in clock hours from the limbus. After 10 days both eyes were processed, half of the group's corneas were assessed histopathologically, and the other half was used for pro/anti-inflammatory cytokine quantification using ELISA. BC treated (Group 1) corneas revealed significantly improved fluorescein staining score for limbal involvement when compared to SS treated (Group 2) corneas at days 4 (p = 0.013), 7 (p < 0.001), and 10 (p < 0.001), respectively. No differences were noted in limbal involvement at day 2 between the two groups (p > 0.99). The overall change (difference in slope) in fluorescein staining for limbal involvement between days 2 and 10 was -0.1669 (p = 0.006). Histologic examinations and cytokine measurements of group 2 demonstrated a strong inflammatory component compared to group 1. Our data indicates that topical application of BC facilitates corneal re-epithelialization and wound healing by suppressing the inflammatory process in an ocular alkali burn model.

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

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

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

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

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.

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.

Protective roles of the TIR/BB-loop mimetic AS-1 in alkali-induced corneal neovascularization by inhibiting ERK phosphorylation.

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.

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.

Cytoprotective effect of crocin and trans-resveratrol on photodamaged primary human retinal pigment epithelial cells.

PURPOSE: Light-induced damage to retinal pigment epithelium during pars plana vitrectomy remains a hot topic in ophthalmology. Improvements in technology led to a change of light sources, selective filters, and shorter light exposure time. Currently, there is no satisfying solution to the problem. The aim of the study was to investigate the cytoprotective effects of crocin and resveratrol on light-induced damage to primary human retinal pigment epithelial cells in vitro. METHODS: Primary human retinal pigment epithelial cells were exposed to light analogous to the illumination during pars plana vitrectomy. To evaluate the cytoprotective effects and potential toxicity of resveratrol and crocin, human retinal pigment epithelial cells were incubated with varying concentrations of both before 3-[4,5-dimethylthiazol-2-yl] tetrazolium bromide (MTT) viability assay. Furthermore, glutathione levels were measured to investigate synergistic antioxidant potential. Apoptosis of human retinal pigment epithelial cells was determined by a nucleosome detection enzyme-linked immunosorbent assay. RESULTS: Crocin and resveratrol improved cell viability in photodamaged human retinal pigment epithelial cells significantly from 40.65 ± 21.99% in illuminated human retinal pigment epithelial cells and reached a peak viability of 85.64 ± 11.37% in crocin and resveratrol pretreated cells (for all: p < 0.001). In line, the combination of the supplements increased glutathione levels significantly from 39.35 ± 21.96% to 80.74 ± 10.32% (p = 0.017). No toxic effects were detected (p > 0.99). However, no change in apoptosis rates could be observed following pretreatment with crocin and resveratrol (p > 0.99). CONCLUSION: Crocin and trans-resveratrol revealed cytoprotective effects on human retinal pigment epithelial cells supporting both supplement's development as potential perioperative treatments in light-induced retinal pigment epithelial damage.

Subconjunctival Injection of Regulatory T Cells Potentiates Corneal Healing Via Orchestrating Inflammation and Tissue Repair After Acute Alkali Burn.

Purpose: This study aimed to investigate the therapeutic effects and underlying mechanisms of locally delivered regulatory T cells (Tregs) on acute corneal wound healing after alkali burn. Methods: After corneal alkali burn, the mice were injected subconjunctivally with regulatory T cells (Tregs) isolated from syngeneic mice. The wound healing process was monitored by clinical manifestation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). As amphiregulin (Areg) was significantly upregulated, its reparative function in injured corneas was suggested. The hypothesis was further verified via loss- and gain-of-function experiments by administrating the antibody of Areg (anti-Areg) and recombinant Areg (rmAreg). Results: Subconjunctivally injected Tregs rapidly migrated to injured corneas. The mice treated with Tregs showed prominently reduced corneal opacity, alleviated edema, and faster re-epithelialization compared with the control group. Mechanistically, Treg treatment led to suppressed infiltration of inflammatory cells, along with improved proliferation and inhibited apoptosis of corneal epithelial cells. Tregs expressed upregulated functional markers, including Areg. Expectantly, the levels of Areg in corneas were dramatically higher in the Treg injection group, in line with better corneal restoration. Additional experiments showed that the administration of anti-Areg blunted the reparative effect of Tregs, while exogenous Areg enhanced it. Treg-treated corneas also exhibited less neovascularization and fibrosis at a later reconstruction stage of corneal repair. Conclusions: The findings showed that the subconjunctival injection of Tregs effectively promoted corneal wound healing by inhibiting excessive inflammation and enhancing epithelial regeneration, with an indispensable reparative role of Areg. Subsequent complications of corneal vascularization and fibrosis were therefore reduced.

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.