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Corrosive esophageal burn (CEB) is a disease with high mortality and morbidity rates. The aim of this study was to determine the efficacy of castor oil, in preventing stricture development at the experimental CEB model. In addition to studying standard histopathological damage data, neopterin, IL-33, and ST-2 proteins were also studied for the first time. Fifty Wistar-Albino rats were divided into randomized 5 groups. [Sham group (G1) (n:10), Control group (G2) (n:10), Early Stage Topical Application group (G3)(n:10), Late Stage Topical Application group (G4) (n:10), Oral Application group (G5) (n:10)]. Weight measurement, esophageal length, histopathological damage score (HDS) and total stenosis score (TSS), tissue caspase-3 and VEGF staining, tissue hydroxyproline (HYP), blood TNF-alpha, IL-6, IL-33, Neopterin, and ST-2 levels were measured. In the castor oil application groups, weight gain was observed, the acute phase reaction decreased, submucosal/tunica muscularis fibrosis (TMF) and muscularis mucosal damage (MMD) were reduced, and TSS and HDS decreased. While no significant difference was detected in the ST-2 protein, which was used for the first time in this study model, a significant increase in neopterin protein was observed in the application groups. Results indicate the nutritional contribution of castor oil, as well as its tissue healing and esophageal stricture-preventing efficacy at histopathological and immune-histochemical levels., (© 2024. The Author(s).)

Purpose: Benzalkonium chloride (BAC) is commonly used as a preservative in ophthalmic medications, despite its potential to induce chemical injury. Extensive research has demonstrated that BAC can lead to adverse effects, including injuries to the ocular surface. Our study aimed to elucidate the underlying mechanism of necroptosis induced by BAC., Methods: Human corneal epithelial (HCE) cells and mouse corneas were subjected to chemical injury, and the necrostatin-1 (Nec1) group was compared to the dimethylsulfoxide (DMSO) group. The extent of damage to HCE cells was assessed using CCK-8 and flow cytometry. Hematoxylin and eosin staining, as well as fluorescein sodium staining, were used to detect and characterize corneal injury. The activation of inflammatory cytokines and necroptosis-related proteins and genes was evaluated using Western blotting, immunofluorescence staining, and quantitative RT‒PCR., Results: In our study, the induction of necroptosis by a hypertonic solution was not observed. However, necroptosis was observed in HCE cells exposed to NaOH and BAC, which activated the receptor-interacting protein kinase 1 (RIPK1) - receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase domain-like protein (MLKL) signaling pathway. In mouse corneal tissues, BAC could induce necroptosis and inflammation. The administration of Nec1 mitigated the inflammatory response and ocular surface damage caused by BAC-induced necroptosis in our experimental models. Furthermore, our in vivo experiments revealed that the severity of necroptosis was greater in the 3-day group than in the 7-day group., Conclusions: Necroptosis plays a role in the pathological development of ocular surface injury caused by exposure to BAC. Furthermore, our study demonstrated that the administration of Nec1 could mitigate the pathological effects of necroptosis induced by BAC in clinical settings., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Topical eye drop approaches to treat ocular inflammation in dry eyes often face limitations such as low efficiency and short duration of drug delivery. Nanofibers serve to overcome the limitation of the short duration of action of topical eye drops used against ocular inflammation in dry eyes. Several attempts to develop suitable nanofibers have been made; however, there is no ideal solution. Here, we developed polycaprolactone (PCL) nanofibers loaded with dexamethasone acetate (DEX), prepared by electrospinning, as a potential ocular drug delivery platform for corneal injury treatment. Thirty-nine Sprague Dawley rats (7 weeks old males) were divided into four treatment groups after alkaline burns of the cornea; negative control (no treatment group); dexamethasone eyedrops (DEX group); PCL fiber (PCL group); dexamethasone loaded PCL (PCL + DEX group). We evaluated therapeutic efficacy of PCL + DEX by examining the epithelial wound healing effect, the extent of corneal opacity and neovascularization. Additionally, various inflammatory factors, including IL-1β, were investigated through immunochemistry, western blot analysis, and quantitative real-time RT-PCR (qRT-PCR). PCL + DEX group showed histologically alleviated signs of corneal inflammation compared with DEX group, which showed a decrease in IL-1β and MMP9 in the corneal stroma. The quantitative expression on day 1 after alkaline burn of pro-inflammatory markers, including IL-1β and IL-6, in the PCL + DEX group was significantly lower than that in the DEX group. Notably, PCL + DEX treatment significantly suppressed neovascularization, and enhanced the anti-inflammatory function of DEX during the acute phase of ocular inflammation. Collectively, these findings suggest that PCL + DEX may be a promising approach to effective drug delivery in corneal burn injuries., (© 2024. The Author(s).)

Ingestion of a corrosive substance may cause corrosive esophagitis. Curcumin has anti-inflammatory and mucosal protective effects. In this study, the effects of curcumin on the acute phase of corrosive esophagitis were investigated. Twenty-seven Wistar Albino rats were divided into four groups; sham (group I), control (group II), and experiment groups (group III, 100 mg/kg curcumin; group IV, 200 mg/kg curcumin). Forty percent sodium hydroxide solution was used to erode the esophagi of rats in groups other than the sham group. Curcumin was applied to animals in the experiment groups 10 min after the corrosion. After 24 h, animals were sacrificed, and esophagus samples were collected. According to the histopathological examination, the muscularis mucosa damage was regressed from 100% in group II to 71.4% in group III and 50% in group IV. Mild level of damage and collagen deposition in the tunica muscularis regressed from 66.7% of the animals in the control group to 42.9% in group III and to none in group IV. Further, an increase in submucosal collagen was present in all samples from groups II and III, while 83.3% of samples had an increase in submucosal collagen in group IV. There was a significant difference in the histopathological total score between the control group and group IV (p=0.02). The results showed that the administration of curcumin in a dose-dependent manner can relieve the acute phase of corrosive esophagitis., (© 2024. The Author(s).)

Purpose: The purpose of this study was to evaluate the safety and efficacy of topical losartan in the therapeutic treatment of established corneal scaring fibrosis at 1 month after alkali burn in rabbits., Methods: Standardized alkali burns were performed in 1 eye of 24 rabbits with 0.75N NaOH for 15 seconds. Corneas were allowed to heal and develop scaring of the cornea for 1 month. Twelve eyes per group were treated with 50 µL of topical 0.8 mg/mL losartan in balanced salt solution (BSS), pH 7.0, and 12 eyes were treated with vehicle BSS 6 times per day. Six corneas were analyzed at 1 week or 1 month in each group. Standardized slit lamp photographs were obtained at the end point for each cornea and opacity was quantitated using ImageJ. Corneoscleral rims were cryofixed in optimum cutting temperature (OCT) solution and combined duplex immunohistochemistry for myofibroblast marker alpha-smooth muscle actin (α-SMA), mesenchymal cell marker vimentin, and TUNEL assay for apoptosis was performed on all corneas., Results: Topical losartan was effective in the treatment of established stromal fibrosis following alkali burn injury to the rabbit cornea. Stromal myofibroblast density was decreased and stromal cell apoptosis was increased (included both α-SMA-positive myofibroblasts and α-SMA-negative, vimentin-positive cells) at both 1 week and 1 month in the topical losartan-treated compared with vehicle-treated groups., Conclusions: Topical losartan is effective in the treatment of established stromal fibrosis in rabbits. Most myofibroblasts disappear from the stroma within the first month of losartan treatment. Longer treatment with topical losartan is needed to allow time for corneal fibroblast regeneration of the epithelial basement membrane (in coordination with epithelial cells) and the removal of disordered extracellular matrix produced by myofibroblasts.

Limbal stem cell deficiency (LSCD) is a clinically challenging eye disease caused by damage to limbal stem cells (LSCs). Currently, the international consensus classifies LSCD into three clinical stages based on the disease severity. However, no existing animal models attempt to replicate the varying degrees of LSCD observed in clinical cases. The present study demonstrates an easy-to-create, reproducible, and reliable mouse model of graded LSCD. To achieve mild, moderate, or severe LSCD, filter paper rings with a variety of central angles (90°, 180°, or 270°) are utilized to deliver alkali burns to different sizes of the limbal area (1, 2, or 3 quarters). The animal model has successfully resulted in the development of clinical signs and pathological manifestations in escalating severity that are similarly observed in the three clinical stages of LSCD. Our study thus provides new insights into distinct pathological features underlying different grades of LSCD and serves as a new tool for further exploring the disease mechanisms and developing new effective therapeutics for repairing damaged LSCs., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The cornea serves as an essential shield that protects the underlying eye from external conditions, yet it remains highly vulnerable to injuries that could lead to blindness and scarring if not promptly and effectively treated. Excessive inflammatory response constitute the primary cause of pathological corneal injury. This study aimed to develop effective approaches for enabling the functional repair of corneal injuries by combining nanoparticles loaded with anti-inflammatory agents and an injectable oxidized dextran/gelatin/borax hydrogel. The injectability and self-healing properties of developed hydrogels based on borate ester bonds and dynamic Schiff base bonds were excellent, improving the retention of administered drugs on the ocular surface. In vitro cellular assays and in vivo animal studies collectively substantiated the proficiency of probucol nanoparticle-loaded hydrogels to readily suppress proinflammatory marker expression and to induce the upregulation of anti-inflammatory mediators, thereby supporting rapid repair of rat corneal tissue following alkali burn-induced injury. As such, probucol nanoparticle-loaded hydrogels represent a prospective avenue to developing long-acting and efficacious therapies for ophthalmic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Severe corneal injury can lead to blindness even after prompt treatment. 14-3-3zeta, a member of an adaptor protein family, contributes to tissue repair by enhancing cellular viability and inhibiting fibrosis and inflammation in renal disease or arthritis. However, its role in corneal regeneration is less studied. In this study, filter disc of 2-mm diameter soaked in sodium hydroxide with a concentration of 0.5 N was placed at the center of the cornea for 30 s to establish a mouse model of corneal alkali injury. We found that 14-3-3zeta, which is mainly expressed in the epithelial layer, was upregulated following injury. Overexpression of 14-3-3zeta in ocular tissues via adeno-associated virus-mediated subconjunctival delivery promoted corneal wound healing, showing improved corneal structure and transparency. In vitro studies on human corneal epithelial cells showed that 14-3-3zeta was critical for cell proliferation and migration. mRNA-sequencing in conjunction with KEGG analysis and validation experiments revealed that 14-3-3zeta regulated the mRNA levels of ITGB1, PIK3R1, FGF5, PRKAA1 and the phosphorylation level of Akt, suggesting the involvement of the PI3K-Akt pathway in 14-3-3zeta-mediated tissue repair. 14-3-3zeta is a potential novel therapeutic candidate for treating severe corneal injury., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Abstract: Cigarette burn lesions present forensic scenarios that are often difficult to investigate, both from a morphological diagnostic point of view and with regard to the mode of infliction, especially if the victim is unable to speak or has died. Although there may be the suspicion for a lesion to be produced by a lit cigarette, to date one can only rely on the morphological aspects that characterize it, and there is a lack of tools to reach the most evidence-based diagnosis possible. This limitation arose when managing a forensic autopsy case of possible child abuse that resulted in the death of the child, characterized by the presence of 3 suspicious cigarette burn lesions. We therefore decided to perform scanning electron microscopy/energy-dispersive x-ray (SEM/EDX) spectrometry analysis on these lesions and on the cigarette butt found at the crime scene. At the same time, SEM/EDX was applied to the analysis of an unlit cigarette in its entirety (obtained from the same source package as the cigarette butt), a positive control skin sample with an iatrogenic cigarette burn injury, and a negative control skin sample. Among the various compounds highlighted on compositional analysis, only sulfuric anhydride (SO 3 ) and phosphoric anhydride (P 2 O 5 ) showed a highly significant distribution pattern by being found in the autopsy samples, the cigarette butt, the tobacco of the unlit cigarette, and the positive skin control. Considering this, cigarette burns appear to follow Locard's principle as well, and similarly to other lesions, SEM/EDX allowed the diagnosis of cigarette burn lesions, already suspected morphologically, to be corroborated. Therefore, SEM/EDX is confirmed as a helpful tool in forensic pathology investigations., Competing Interests: Conflicts of interest/competing interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Alkaline burns to the cornea lead to loss of corneal transparency, which is essential for normal vision. We used a rat corneal alkaline burn model to investigate the effect of ophthalmic trimebutine solution on healing wounds caused by alkaline burns. Trimebutine, an inhibitor of the high-mobility group box 1-receptor for advanced glycation end products, when topically applied to the burned cornea, suppressed macrophage infiltration in the early phase and neutrophil infiltration in the late phase at the wound site. It also inhibited neovascularization and myofibroblast development in the late phase. Furthermore, trimebutine effectively inhibited interleukin-1β expression in the injured cornea. It reduced scar formation by decreasing the expression of type III collagen. These findings suggest that trimebutine may represent a novel therapeutic strategy for corneal wounds, not only through its anti-inflammatory effects but also by preventing neovascularization., (© 2024. The Author(s).)

Burn as physical injury ranks as the fourth most prevalent trauma across the world. In this study, we aimed to compare the impact of gasoline burn and chromic acid burn on the internal organs and immune functions in rats. The results showed that the levels of methemoglobin (MHb) to total hemoglobin (Hb) as well as the Cr6+ content showed significant elevation in the chromic acid burn group relative to the gasoline burn group. HE staining was used to evaluate the histological changes in the injured tissues as well as the tissues excised from internal organs. We found that chromic acid burn-induced more severe damage to rat tissues. Gasoline burn showed no significant impact on the intestinal tissues of rats, while the chromic acid burn-induced increased cell death in rat intestines. Moreover, the results of HE staining also revealed that gasoline burn and chromic acid burn showed no evident impact on rat hearts. Gasoline burn also showed no significant effects on the liver, lungs and kidneys of rats, while the chromic acid burn caused injuries to such internal organs in comparison with the control and gasoline burn groups. In addition, the MPO activity was higher in the liver, intestine, lungs and kidneys of rats with chromic acid burn. Furthermore, the expression of inflammation response cytokines was examined in the serum of rats. The results demonstrated that the levels of IL-6, IL-1β and TNF-α showed a significant increase in both the gasoline burn and chromic acid burn groups of rats relative to the control, and the levels were higher in the chromic acid burn group in comparison with the gasoline burn group. In conclusion, the chromic acid burn-induced more severe organ injury, inflammation and immune response compared with the gasoline burn, which may provide reference data for the clinical treatment of patients with different burn injuries.

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Caustic ingestion is associated with long-term sequelae like esophageal stricture, gastric cicatrization, and long-term risk of dysplasia or even carcinoma. However, only a few small studies have explored histopathological aspects of caustic-induced esophageal/gastric injury., Materials and Methods: We retrospectively evaluated specimens of patients undergoing surgery due to caustic ingestion-related complications from 2008 to 2020. Pathological examination was conducted by two independent gastro-pathologists to evaluate the extent and depth of the caustic injury, presence or absence of tissue necrosis, type and degree of inflammation, or presence of any dysplastic cells., Results: A total of 54 patients underwent surgical exploration during the inclusion period and complete details of 39 specimens could be retrieved. The mean age of the included patients was 28.66 ± 9.31 years and 25 (64.1%) were male. The majority of patients (30; 76.9%) had a history of caustic ingestion more than three months before the surgery and the presence of long or refractory stricture was the most common indication for the surgery (20; 51.28%). In the resected specimen, a majority of patients had superficial esophageal or gastric ulcer (90.6%; 60.0%), transmural inflammation (68.8%; 65.6%), transmural fibrosis (62.5%; 34.4%), and hypertrophied muscularis mucosa (78.13%; 53.3%). However, none of the patients had dysplasia in the resected esophageal or gastric specimens., Conclusion: Caustic ingestion leads to mucosal ulceration, transmural inflammation, and transmural fibrosis which might be the reason for refractory stricture in such patients., (Copyright © 2024 Copyright: © 2024 Indian Journal of Pathology and Microbiology.)

Objective: To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization., Methods: The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 μM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at －80 ℃ until histological study or protein extraction., Results: Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC., Conclusion: This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.

Creating of a scar model in laboratory animals is the most acceptable option for the preclinical search of scar treatment. However, due to high skin regeneration rate in laboratory rodents, creating an optimal animal model of scar formation is a challenge. Here we describe five methods for modeling a scar tissue in rats that we have tested. These methods allowed achieving different histopathological features and different stages of skin scar formation., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Corneal alkali burns can lead to ulceration, perforation, and even corneal blindness due to epithelial defects and extensive cell necrosis, resulting in poor healing outcomes. Previous studies have found that chitosan-based in situ hydrogel loaded with limbal epithelium stem cells (LESCs) has a certain reparative effect on corneal alkali burns. However, the inconsistent pore sizes of the carriers and low cell loading rates have resulted in suboptimal repair outcomes. In this study, 4D bioprinting technology was used to prepare a chitosan-based thermosensitive gel carrier (4D-CTH) with uniform pore size and adjustable shape to improve the transfer capacity of LESCs., Methods: Prepare solutions of chitosan acetate, carboxymethyl chitosan, and β-glycerophosphate sodium at specific concentrations, and mix them in certain proportions to create a pore-size uniform scaffold using 4D bioprinting technology. Extract and culture rat LESCs (rLESCs) in vitro, perform immunofluorescence experiments to observe the positivity rate of deltaNp63 cells for cell identification. Conduct a series of experiments to validate the cell compatibility of 4D-CTH, including CCK-8 assay to assess cell toxicity, scratch assay to evaluate the effect of 4D-CTH on rLESCs migration, and Calcein-AM/PI cell staining experiment to examine the impact of 4D-CTH on rLESCs proliferation and morphology. Establish a severe alkali burn model in rat corneas, transplant rLESCs onto the injured cornea using 4D-CTH, periodically observe corneal opacity and neovascularization using a slit lamp, and evaluate epithelial healing by fluorescein sodium staining. Assess the therapeutic effect 4D-CTH-loaded rLESCs on corneal alkali burn through histological evaluation of corneal tissue paraffin sections stained with hematoxylin and eosin, as well as immunofluorescence staining of frozen sections., Results: Using the 4D-CTH, rLESCs were transferred to the alkali burn wounds of rats. Compared with the traditional treatment group (chitosan in situ hydrogel encapsulating rLESCs), the 4D-CTH-rLESC group had significantly higher repair efficiency of corneal injury, such as lower corneal opacity score (1.2 ± 0.4472 vs 0.4 ± 0.5477, p < 0.05) and neovascularization score (5.5 ± 1.118 vs 2.6 ± 0.9618, p < 0.01), and significantly higher corneal epithelial wound healing rate (72.09 ± 3.568% vs 86.60 ± 5.004%, p < 0.01)., Conclusion: In summary, the corneas of the 4D-CTH-rLESC treatment group were similar to the normal corneas and had a complete corneal structure. These findings suggested that LESCs encapsulated by 4D-CTH significantly accelerated corneal wound healing after alkali burn and can be considered as a rapid and effective method for treating epithelial defects., (© 2024. The Author(s).)

Toilet cleaner containing hydrochloric acid is a common item found in households all over the world. Due to the availability of the substance, it becomes one of the main contributors to corrosive damage to the gastrointestinal system. This study reports a case of a female in her 50s with an alleged history of ingestion of toilet cleaner an empty bottle of which was found together with a suicide note at the incident site. During the autopsy, the forensic expert made an intriguing observation regarding the dispersion of ingested acid to other organs without gastric perforation. Despite the absence of gastric perforation, the corrosive effects of the ingested acid were evident in various organs, including the liver and spleen. This phenomenon suggests a unique mechanism by which the acid is able to disperse and cause damage beyond the stomach, leading to widespread organ involvement. However, through a comprehensive analysis of the detailed history, typical macroscopic autopsy findings, and chemical analysis reports, it is possible to establish that the cause of death is corrosive acid poisoning. In such cases, further investigation is warranted to gain a better understanding of the underlying mechanisms responsible for the dispersion of the acid and its clinical implications. By delving deeper into these aspects, we can enhance our knowledge and contribute to the field of forensic medicine., (Copyright © 2024 by PTMSiK.)

Laser Doppler imaging (LDI) technology has been validated to assess thermal burn depth by predicting wound healing potential. However, there is no clear evidence for its use in chemical burns. We present a case of an 8% total burn surface area (TBSA) nitric acid burn following an industrial accident, in an otherwise healthy 36-year-old man. LDI assessment was suggestive of poor healing potential of >21 days, warranting surgical management. However, conservative management was opted for based on clinical assessment as the wound eschar appeared thin and more consistent with epithelial staining. Patient follow-up confirmed a total burn healing time of two months, suggesting that the LDI assessment was accurate. A comprehensive literature review was performed using the MEDLINE (PubMed) database to identify animal or clinical studies evaluating the efficacy of LDI in chemical burns. A qualitative synthesis of our findings is presented. We identified two experimental studies in porcine models with sulfur mustard burns, each confirming the accuracy of LDI assessment when compared to the histopathology findings. Limited experimental animal studies on the use of LDI suggest similar validity in chemical burns, and this correlates with the clinical outcome in this case. However, this alone is insufficient to prove its validity and define its role in the assessment of chemical burns. Clinical trials are required to further assess and define the parameters of LDI use and efficacy in this context., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Burn Association.)

Gallic acid, acting as an antioxidant, anti-precipitant and cytoprotective agent, was used as a possible remedial natural component for treating experimentally induced esophageal burn. Wistar rats (n=24) were divided into three groups. Control group was given 1 mL 0.9% NaCl. Experimental esophageal burn was induced with 1 mL 40% NaOH application to the esophagus in groups 2 and 3. Gallic acid® (20 mg/kg) was administered to the treated group via oral gavage for 10 days. Removed tissues were fixed and paraffin blocks were prepared. Histopathological examination was performed after the sections had been stained with hematoxylin-eosin. Tumor necrosis factor alpha and caspase-3 antibodies were used on immunohistochemical analysis. In the esophageal burn group, necrosis, degeneration and numerous apoptotic cells, as well as intense inflammatory cell infiltration and fibrosis in the muscle layer were observed under light microscope. In the treated group, remodeling of epithelial cells with marked reduction in the connective tissue collagen content was observed, as well as marked reduction in the volume of collagen and abundance of inflammatory cells in blood vessels. Gallic acid treatment may help heal esophageal burns and prevent complications., (Sestre Milosrdnice University Hospital.)

Background: Corneal neovascularization (CNV) can be caused by chemical burns. Macrophages are involved in angiogenesis and lymphangiogenesis during CNV. The aim of this study was to investigate whether Wilms' tumor 1-associated protein (WTAP) is involved in macrophage recruitment and VEGF secretion via N6-methyladenosine (m6A) modification., Methods: A CNV mouse model was established by corneal alkali burn. Tumor necrosis factor alpha (TNF-α) was used to stimulate vascular endothelial cells. m6A immunoprecipitation qPCR was used to determine the enrichment of m6A levels in mRNAs. The H3K9me3 enrichment in the promoter region of CC motif chemokine ligand 2 (CCL2) was detected by chromatin immunoprecipitation assay. The WTAP inhibition in vivo was performed using the adeno-associated virus., Results: In the alkali burn corneal tissues, angiogenesis and lymphangiogenesis were promoted as CD31 and LYVE-1 expressions were elevated, and the number of macrophages as well as WTAP expression were increased. Under the TNF-α stimulation, WTAP promoted the recruitment of endothelial cells to macrophages by promoting CCL2 secretion. Mechanistically, WTAP affected the enrichment of H3K9me3 at the CCL2 promoter by regulating the m6A level of SUV39H1 mRNA. The in vivo experiment showed that VEGFA/C/D secretion of macrophages was reduced after WTAP interference. Mechanistically, WTAP regulated the translational efficiency of HIF-1α via m6A modification., Conclusion: WTAP affected macrophage recruitment to endothelial cells via regulation of H3K9me3-mediated CCL2 transcription. WTAP also affected macrophage secretion of VEGFA/C/D via m6A-mediated translation regulation of HIF-1α. Both pathways were involved in the WTAP regulation of angiogenesis and lymphangiogenesis during CNV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus and adversely affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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-1β, 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background and Objectives: Ocular alkaline burn is a clinical emergency that can cause permanent vision loss due to limbal stem cell deficiency and corneal neovascularization (CNV). Although the basic pathogenetic mechanisms are considered to be acute oxidative stress and corneal neovascularization triggered by inflammation, the underlying intracellular mechanisms have not been clearly elucidated. The aim of this study was to investigate the role of endoplasmic reticulum (ER) stress on inflammation and neovascularization, and the effect of the ER stress inhibitor salubrinal (SLB), as a novel treatment in a corneal alkaline burn model in rats. Methods: Chemical burns were created by cautery for 4 s using a rod coated with 75% silver nitrate and 25% potassium nitrate in the corneal center for the corneal neovascularization (CNV) model. Twenty-eight Wistar albino rats were divided into four groups: SHAM, CNV, CNV + SLB, and CNV + bevacizumab (BVC). After the CNV model was applied to the right eye, a single subconjunctival dose (0.05 mL) of 1 mg/kg salubrinal was injected into both eyes in the CNV + SLB group. A total of 1.25 mg/mL of subconjunctival BVC was administered to the CNV + BVC group. Fourteen days after experimental modeling and drug administration, half of the globes were placed in liquid nitrogen and stored at -20 °C until biochemical analysis. The remaining tissues were collected and fixed in 10% buffered formalin for histopathological and immunohistochemical analysis. Three qualitative agents from three different pathways were chosen: TNFR for inflammation, endothelial nitric oxide synthase (e-NOS) for vascular endothelial growth factor (VEGF)-mediated vascular permeability, and caspase-3 for cellular apoptosis. Results: Significantly lower caspase-3 and eNOS levels were detected in the CNV + SLB and CNV + BVC groups than in the CNV group. Additionally, histopathological evaluation revealed a significant decrease in neovascularization, inflammatory cell infiltration, and fibroblast activity in the CNV + SLB and CNV + BVC groups. The endoplasmic reticulum stress inhibitor, salubrinal, administered to the treatment group, attenuated apoptosis (caspase-3) and inflammation (e-NOS). In the control group (left eyes of the SLB group), salubrinal did not have a toxic effect on the healthy corneas. Conclusion: The ER stress pathway plays an important role in angiogenesis after alkaline corneal burns, and treatment with SLB modulates this pathway, reducing caspase-3 and eNOS levels. Further studies are needed to understand the molecular mechanisms altered by SLB-mediated therapy. The fact that more than one mechanism plays a role in the pathogenesis of CNV may require the use of more than one molecule in treatment. SLB has the potential to affect multiple steps in CNV pathogenesis, both in terms of reducing ER stress and regulating cellular homeostasis by inhibiting the core event of integrated stress response (ISR). Therefore, it can be used as a new treatment option and as a strengthening agent for existing treatments. Although blockade of intracellular organelle stress pathways has shown promising results in experimental studies, more in-depth research is needed before it can be used in routine practice. To the best of our knowledge, this study is the first to report the role of ER stress in corneal injury.

Water irrigation is an efficacious decontaminating method for dermis exposures to corrosive agents and hence has been widely applied to treat especially alkali burns. Nevertheless, once alkali has infiltrated the deep subcutaneous tissue, washing the tissue surface with water irrigation does not attenuate the damage progress. Therefore, significant efforts have been devoted to promising strategies aimed at removing the deeply infiltrated lye. According to a recent report, the negative pressure wound therapy (NPWT) reduces the pH value of the exudate from alkali-provoked burns thus accelerating wound healing. However, it remains to be ascertained whether or not NPWT coupled with water irrigation, that is, iNPWT, more effectively hinders the alkali injury deepening. In this study, we compared the effectiveness of an early application of water irrigation with or without NPWT in preventing the progressive deepening of the alkali burn in an animal model. Our histological examination results showed no appreciable difference in tissue injury depth, dermal retention, inflammatory cell infiltration, re-epithelization, and cellular function between iNPWT and water irrigation alone treatments. Thus, our results prove that the more expensive NPWT coupled with water irrigation does not more effectively hinder the alkali's injury deepening. Hence, iNPWT use should be more cautious in clinical practice., (© 2022 The Authors. International Wound Journal published by Medicalhelplines.com Inc (3M) and John Wiley & Sons Ltd.)

An array of corneal pathologies collectively called mustard gas keratopathy (MGK) resulting from ocular exposure to sulfur mustard (SM) gas are the most prevalent chemical warfare injury. MGK involves chronic ocular discomfort that results in vision impairment. The etiology of MGK remains unclear and poorly understood primarily due to a lack of scientific data regarding structural and cellular changes in different layers of the cornea altered by mustard vapor exposure in vivo. The goals of this study were to (a) characterize time-dependent changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ by employing state-of-the-art multimodal clinical ophthalmic imaging techniques and (b) determine if SM-induced acute changes in corneal cells could be rescued by a topical eye drop (TED) treatment using in an established rabbit in vivo model. Forty-five New Zealand White Rabbit eyes were divided into four groups (Naïve, TED, SM, and SM + TED). Only one eye was exposed to SM (200 mg-min/m 3 for 8 min), and each group had three time points with six eyes each (Table-1). TED was topically applied twice a day for seven days. Clinical eye examinations and imaging were performed in live rabbits with stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, and applanation tonometry were conducted to measure corneal haze, ocular surface aberrations, tears, and intraocular pressure respectively. H&E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of corneal epithelium, stroma, and endothelium in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naïve cornea. Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Neither naive nor TED-alone treated eyes showed any structural, cellular, and functional abnormalities. Topical TED treatment significantly reduced SM-induced abnormalities in primary corneal layers. We conclude that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo, and efficient targeting of them with suitable agents has the potential to mitigate SM ocular injury., (Copyright © 2022. Published by Elsevier Ltd.)

The cornea, the transparent part of the eye, performs a significant function in eyesight by refracting the light to focus a visual image. Since the cornea is indispensable for vision, corneal inflammation may induce visual disturbance and blindness. Several investigations have reported that various corneal inflammatory diseases cause visual impairment and chronic inflammation of the cornea, which can lead to blindness. The present study aimed to assess the effect of adipose-derived mesenchymal stem cells (ADMSCs) on corneal healing after alkali injuries. Corneal alkali injuries were induced in the eyes of 20 rabbits. The MSC group (n=10) was treated with subconjunctival injections, while the control group (n=10) was left without any treatment. Rabbits underwent slit-lamp examination and photography and were evaluated for corneal neovascularization. Based on the histological evaluation, the eyes treated with MSCs showed better recovery. Furthermore, the MSC and control groups were significantly different in the degree of corneal neovascularization and re-epithelialization, as well as the elevation of the neovascular tissue at two and four weeks post-surgery., Competing Interests: The authors declare that they have no conflict of interest.

Pathological features of alkali concentration-associated burn were studied using non-invasive anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA). Alkali burn was induced in C57BL/6J mice (n = 20) by placing filter paper soaked in 0.1, 0.25, 0.5, and 1 M NaOH for 30s on the right eye (left eye control). Longitudinal imaging was performed with AS-OCT/OCTA and fluorescein angiography over 14 days, after which eyes were enucleated at 7 and 14 days for histology and immunofluorescence. Concentration-associated corneal swelling was maximal at 0.5M, increasing linearly in a concentration-dependent fashion at 0.1, 0.25, and 0.5 M NaOH, to levels of 50%, 100%, and 175% of control, respectively. At 0.1M, corneal swelling and surface erosions were prominent, while at 0.25M, deep tissue damage, limbal neovascularization, and stromal haze were evident at 7 days. At 0.5M and 1M, severe exacerbation of the corneal swelling, angle closure, Descemet's membrane detachment, hyphema, and profuse central neovascularization were noted as early as day 3, which further progressed to inflammation, fibrosis, and opacity by day 7. We conclude that alkali concentration-dependent burn intensity biomarkers can be assessed by non-invasive AS-OCT/OCTA, distinguishing between mild, moderate, and severe ocular injury, with potential relevance toward clinical utilization in human eyes., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: Retinal ganglion cell (RGC) apoptosis is one of the most severe complications that causes permanent visual impairment following ocular alkali burn (OAB). Currently, very few treatment options exist for this condition. This study was conducted to determine the effect of 4-phenylbutyric acid (4-PBA) on endoplasmic reticulum (ER) stress after OAB using a well-established OAB mouse model., Methods: Ocular alkali burn was induced in C57BL/6 mouse corneas using 1 M NaOH. 4-PBA (10 mg/kg; 250 μL per injection) or saline (250 μL per injection) was injected intraperitoneally once per day for 3 days before the establishment of the OAB model. The apoptosis of retinal ganglion cells (RGCs) was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and the histological damage was examined by hematoxylin and eosin and immunofluorescence assay on retinal flat mounts. The key inflammatory response and the expression of ER stress-related markers in the retinal tissues were assessed by real-time PCR, western blotting and histologic analyses., Results: 4-PBA significantly alleviated the apoptosis of RGCs and prevented the structural damage of the retina, as determined by the evaluation of RGC density and retinal thickness. Inhibition of ER stress by 4-PBA decreased the expression of vital proinflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 beta; and suppressed the activation of retinal microglial cells and nuclear factor-kappa B (NF-κB). 4-PBA reduced the expression of the ER stress molecules, glucose-regulated protein 78, activated transcription factor 6, inositol-requiring enzyme-1 (IRE1), X-box-binding protein 1 splicing, and CCAAT/enhancer-binding protein homologous protein, in the retinal tissues and RGCs of OAB mice., Conclusions: The present study demonstrated that the inhibition of ER stress by 4-PBA alleviates the inflammatory response via the IRE1/NF-κB signaling pathway and protects the retina and RGCs from injury in an OAB mouse model. Such findings further suggest that 4-PBA might have potential therapeutic implications for OAB treatment., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Purpose: To describe the histopathological characteristics of limbal stem cell deficiency (LSCD) due to chronic vernal keratoconjunctivitis (VKC)., Methods: This retrospective study included 14 eyes of 13 patients who underwent simple limbal epithelial transplantation for total LSCD from 2017 to 2018. The histological characteristics of the excised fibrovascular pannus were compared between 2 groups of 7 eyes, each with LSCD due to VKC and chemical burns (CB). Histological characteristics and type of inflammation were studied using special stains and immunohistochemistry. Fisher exact test was used to detect the statistical significance of the histological differences between both groups., Results: Epithelial hypertrophy, epithelial downgrowth, and eosinophilic infiltration were noted in all eyes in the VKC group (7/7, 100%). Epithelial hypertrophy was noted in 3 of the 7 (42.8%) eyes in the CB group, whereas epithelial downgrowth and eosinophilic infiltrates were absent. The average chronic inflammatory score of the pannus (5.28) was higher in VKC than in CB (3.85; P = 0.1080). The presence of goblet cells was higher in the CB group (5/7, 1.4%) than in the VKC group (3/4, 2.8%), although not statistically significant. Other histological differences between the groups were not statistically significant., Conclusions: The histopathological features of LSCD in VKC reveal some distinctive characteristics. These include the presence of epithelial downgrowth, eosinophilic infiltration, and epithelial solid and cystic implants. Although this information may be used to establish the diagnostic criteria for VKC as the cause of LSCD, further studies are needed to elucidate the reasons behind these unique findings., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Aim: This study aimed to investigate the efficiency of topically applied pycnogenol (PYC) in healing the standardized alkaline corneal ulcer in diabetic and normal rats., Materials and Methods: The corneal alkali-burn injury (CA-I) model was unilaterally developed in Wistar rats by filter paper saturated with 0.01 M of NaOH and touching the eyes for 45 s. Rats were divided into four groups: Normal control (NC), normal PYC (NPYC), diabetic control (DC), and diabetic PYC (DPYC). Both NPYC and DPYC groups were daily treated with PY eye drops three times, whereas NC and DC ones were treated with ordinary saline for six successive days., Results: The wound healing of corneal epithelial was improved in the NPYC group compared to the NC group. Meanwhile, it was significantly improved (P < 0.05) in the DPYC group than in the DC group. Histological examination revealed that corneal re-epithelialization was more accomplished in the DPYC group than in the DC group. In addition, the inflammatory cells were augmented in the DC group more than those in the DPYC one., Conclusion: The findings obtained revealed the efficiency of PYC for enhancing the corneal re-epithelialization and reducing the inflammatory reaction post alkali burn in rats, and thus it could be beneficially valuable as a treatment for the diabetic keratopathy., (© 2022. The Author(s).)

Background: Apoptosis signal-regulating kinase 1-interacting protein 1 (AIP1) participates in inflammatory neovascularization induction. NADPH oxidase 4 (NOX4) produces reactive oxygen species (ROS), leading to an imbalance in nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) and NLR family pyrin domain containing 6 (NLRP6) expression. The mechanisms of AIP1, NOX4, ROS and inflammasomes in corneal neovascularization were studied herein., Methods: C57BL/6 and AIP1-knockout mice were used in this study. The alkali burn procedure was performed on the right eye. Adenovirus encoding AIP1 plus green fluorescence protein (GFP) (Ad-AIP1-GFP) or GFP alone was injected into the right anterior chamber, GLX351322 was applied as a NOX4 inhibitor, and then corneal neovascularization was scored. The expression of related genes was measured by quantitative real-time polymerase chain reaction, western blotting and immunofluorescence staining. 2',7'-Dichlorofluorescin diacetate staining was used to determine the ROS levels., Results: The expression of AIP1 was decreased, while that of cleaved interleukin-1β (clv-IL-1β) and vascular endothelial growth factor A (VEGFa) was increased after alkali burn injury. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. NLRP3/NLRP6 expression was imbalanced after alkali burns. GLX351322 reversed the imbalance in NLRP3/NLRP6 by reducing the ROS levels. This treatment also reduced the expression of clv-IL-1β and VEGFa, suppressing neovascularization., Conclusions: AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burn injury. Based on the pathogenesis of corneal neovascularization, these findings are expected to provide new therapeutic strategies for patients. Corneal alkali burn injury is a common type of ocular injury that is difficult to treat in the clinic. The cornea is a clear and avascular tissue. Corneal neovascularization after alkali burn injury is a serious complication; it not only seriously affects the patient's vision but also is the main reason for failed corneal transplantation. Corneal neovascularization affects approximately 1.4 million patients a year. We show for the first time that AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burns. The expression of AIP1 was decreased, while that of clv-IL-1β and VEGFa was increased after alkali burns. We tried to elucidate the specific molecular mechanisms by which AIP1 regulates corneal neovascularization. NOX4 activation was due to decreased AIP1 expression in murine corneas with alkali burns. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. Additionally, NLRP3/NLRP6 expression was unbalanced, with NLRP3 activation and NLRP6 suppression in the corneal alkali burn murine model. Eye drops containing GLX351322, a NOX4 inhibitor, reversed the imbalance in NLRP3/NLRP6 by reducing ROS expression. This treatment also reduced the expression of clv-IL-1β and VEGFa, reducing neovascularization. Therefore, we provide new gene therapeutic strategies for patients. With the development of neovascularization therapy, we believe that in addition to corneal transplantation, new drug or gene therapies can achieve better results. Video Abstract., (© 2022. The Author(s).)

Aim/background: Esophageal strictures in children that develop as a result of accidental ingestion of corrosive substances remain an important health problem. The purpose of this study is to determine the effects of Hesperidin, an effective bioflavonoid in the proliferative and exudative phase of inflammation, on the stricture formation in corrosive esophageal burns., Methods: Experimental esophageal burns in rats were created using a modified Gehanno and Guedon model with 20% NaOH. Rats were divided into 5 groups. In the Sham group, the distal esophagus was prepared and cannulated according to the model, but no NaOH was administrated. The esophageal burn was created with NaOH in the other groups. The burned groups were divided into two groups as untreated (T 14 , T 21 ) and treated with 100 mg/kg/day Hesperidin (H 14 , H 21 ) intraperitoneally, and these groups were divided into two according to their sacrification periods (14 and 21 days). Inflammation, fibrosis, and necrosis were graded by histopathological evaluation in all groups. The efficacy of treatment was evaluated using the weight of rats, stenosis index, and histopathological parameters., Results: Histopathologic damage scores such as inflammation, necrosis, and fibrosis were lower in the H 14 and H 21 groups and higher in the T 14 and T 21 groups. And also stenosis index was found higher in T 14 and T 21 groups (p < 0.05), while it was similar to the Sham group in H 14 and H 21 groups. No statistically significant difference was found between the H 14 and H 21 groups in terms of stenosis index. When weights of the rats at the beginning and end of the experiment were compared, weights of the H 14 and H 21 groups and the Sham group were similar. There was a significant decrease in the weight of the rats in the T 14 and T 21 groups (p < 0.001)., Conclusions: This study is the first to use Hesperidin in preventing esophageal damage in an esophageal caustic burn model. It was shown that Hesperidin was effective in reducing macroscopic and microscopic histopathologic damage in the corrosive esophageal burn model, preventing the stricture formation, and has positive effects on nutrition in rats with an esophageal burn., (© 2021. The Japan Esophageal Society.)

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)-1β, IL-1β, 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-1β, pro-IL-18, IL-1β 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

Introduction: Alcohol-based hand sanitizers (ABHS) are widely used for hand hygiene due to the coronavirus disease pandemic. However, risk awareness regarding its adverse effects is lacking. We aim to report a case of ocular chemical burn that showed severe clinical presentation associated with ABHS., Patient Concerns: A 5-year-old girl presented with severe left eye pain after 62% gel-type ABHS splashed into her eye., Diagnosis: On slit lamp examination, a near total corneal and conjunctival epithelial defect with limbal pale on the lower half of the cornea was noted. Severe ocular burn by ABHS was prominent with suspected limbal stem cell damage., Interventions: She was hospitalized and was prescribed topical medications including antibiotics, steroid eye drops with preservative-free artificial tears, and oral nonsteroidal anti-inflammatory drugs., Outcomes: Despite intensive medical treatments, the corneal and conjunctival epithelial defects showed no improvement up to the 4th hospital day. After additional instillation of autoserum eye drops to promote epithelial healing, the corneal epithelium barely recovered from the temporal limbus. On the third week of admission, the epithelial defect was completely resolved without corneal opacity, although with minimal symblepharon in the lower fornix., Conclusions: Gel-type ABHS can cause severe form of ocular chemical burn such as delayed ocular surface healing. In clinical setting, immediate and thorough rinsing of alcohol-based gel and early intensive treatment are crucial., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: To test whether an acute corneal injury activates a proinflammatory reflex, involving corneal sensory nerves expressing substance P (SP), the hypothalamus, and the sympathetic nervous system., Methods: C57BL6/N (wild-type [WT]) and SP-depleted B6.Cg-Tac1tm1Bbm/J (TAC1-KO) mice underwent bilateral corneal alkali burn. One group of WT mice received oxybuprocaine before alkali burn. One hour later, hypothalamic neuronal activity was assessed in vivo by magnetic resonance imaging and ex vivo by cFOS staining. Some animals were followed up for 14 days to evaluate corneal transparency and inflammation. Tyrosine hydroxylase (TH), neurokinin 1 receptor (NK1R), and neuronal nitric oxide synthase (nNOS) expression was assessed in brain sections. Sympathetic neuron activation was evaluated in the superior cervical ganglion (SCG). CD45+ leukocytes were quantified in whole-mounted corneas. Noradrenaline (NA) was evaluated in the cornea and bone marrow., Results: Alkali burn acutely induced neuronal activation in the trigeminal ganglion, paraventricular hypothalamus, and lateral hypothalamic area (PVH and LHA), which was significantly lower in TAC1-KO mice (P < 0.05). Oxybuprocaine application similarly reduced neuronal activation (P < 0.05). TAC1-KO mice showed a reduced number of cFOS+/NK1R+/TH+ presympathetic neurons (P < 0.05) paralleled by higher nNOS expression (P < 0.05) in both PVH and LHA. A decrease in activated sympathetic neurons in the SCG and NA levels in both cornea/bone marrow and reduced corneal leukocyte infiltration (P < 0.05) in TAC1-KO mice were found. Finally, 14 days after injury, TAC1-KO mice showed reduced corneal opacity and inflammation (P < 0.05)., Conclusions: Our findings suggest that stimulation of corneal sensory nerves containing SP activates presympathetic neurons located in the PVH and LHA, leading to sympathetic activation, peripheral release of NA, and corneal inflammation.

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-1β, 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., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-β (TGF-β) 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.

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.)

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.

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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-1β 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-1β 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-1β, and Cxcr2 expression after CAB. These candidate lncRNAs and mRNAs may be the potential targets for the treatment strategy of the alkali injured cornea.

Alkali burn to the cornea is one of the most intractable injuries to the eye due to the opacity resulting from neovascularization (NV) and fibrosis. Numerous studies have focused on studying the effect of drugs on alkali-induced corneal injury in mouse, but fewer on the involvement of alkali-induced DNA methylation and the PI3K/AKT/mTOR signaling pathway in the mechanism of alkali-induced corneal injury. Thus, the aim of this study was to determine the involvement of DNA methyltransferase 3 B-madiated DNA methylation and PI3K/AKT/mTOR signaling modulation in the mechanism of alkali-induced corneal injury in a mouse model. To this end, we used bisulfite sequencing polymerase chain reaction and Western blot analysis, to study the effects of 5-aza-2'-deoxycytidine and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, which inhibit methyltransferase and PI3K respectively, on DNA methylation and expression of downstream effectors of PI3K related to corneal NV, including TSC1 and mTOR genes. The results showed that, after an intraperitoneal injection of rapamycin (2 mg/kg/day) for seven days, the alkali-induced opacity and NV were remarkably decreased mainly by suppressing the infiltration of immune cells into injured corneas, angiogenesis, VEGF expression and myofibroblasts differentiation; as well as by promoting corneal cell proliferation and PI3K/AKT/mTOR signaling. More significantly, these findings showed that epigenetic regulatory mechanisms by DNA methylation played a key role in corneal NV, including in corneal alkali burn-induced methylation modification and rapamycin-induced DNA demethylation which involved the regulation of the PI3K/AKT/mTOR signaling pathway at the protein level. The precise findings of morphological improvement and regulatory mechanisms are helpful to guide the use of rapamycin in the treatment of corneal angiogenesis induced by alkaline-burn., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.