The Potential Reversible Transition between Stem Cells and Transient-Amplifying Cells: The Limbal Epithelial Stem Cell Perspective.

Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs.

Central toxic keratopathy leading to epithelial ingrowth following femtosecond LASIK.

We report a case of a woman in her 30s who underwent femtosecond LASIK (laser-assisted in situ keratomileusis) in both eyes to correct her simple myopic astigmatism. After the surgery, both eyes developed diffuse lamellar keratitis, and intensive topical steroids were initiated to control the same. Subsequently, central toxic keratopathy (CTK) developed bilaterally. Three weeks after the surgery, the right eye showed signs of progressive epithelial ingrowth involving the pupillary area. Surgical intervention in the form of flap relift followed by debridement of the epithelial cells and an alcohol interface wash were performed to treat the same. This is the first report of an epithelial ingrowth following CTK after femtosecond LASIK.

A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery.

Purpose: The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Methods: Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). Results: The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3 days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. Conclusions: These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. Translational Relevance: The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

Purpose: In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Methods: Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Results: Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. Conclusions: ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

Impact of Obesity and Age on Mouse Corneal Innervation at the Epithelial-Stromal Interface.

Purpose: The corneal epithelium is the most highly innervated structure in the body. Previously, we reported a novel event whereby stromal axons fuse with basal epithelial cells, limiting nerve penetration into the epithelium. Although corneal-epithelial nerves undergo changes in sensitivity and distribution throughout life and in response to an obesogenic diet, it is unknown if neuronal-epithelial cell fusion is altered. Here, we sought to determine if neuronal-epithelial cell fusion frequency correlates with obesogenic diet consumption and age. Methods: Corneas were collected from C57BL/6 mice and evaluated for neuronal-epithelial cell fusion frequency using serial block-face scanning electron microscopy. To assess the correlation between diet-induced obesity and fusion frequency, 6-week-old mice were fed either a normal diet or an obesogenic diet for 10 weeks. To assess changes in fusion frequency between young and adult mice under normal dietary conditions, 9- and 24-week-old mice were used. Results: Mice fed a 10-week obesogenic diet showed 87% of central-cornea stromal nerves engaged in fusion compared with only 54% in age-matched controls (16 weeks old). In 9-week-old normal-diet animals, 48% of central-cornea stromal nerves contained fusing axons and increased to 81% at 24 weeks of age. Corneal sensitivity loss correlated with increased body weight and adiposity regardless of age and diet. Conclusions: Neuronal-epithelial cell fusion positively correlates with age and obesogenic diet consumption, and corneal nerve sensitivity loss correlates with increased body weight and adiposity, regardless of age and diet. As such, neuronal-epithelial cell fusion may play a role in corneal nerve density and sensitivity regulation.

Uneven Corneal Epithelial Redistribution After Femtosecond Laser-Assisted Lenticule Intrastromal Keratoplasty in Correcting Moderate-to-High Hyperopia.

PURPOSE: To evaluate the characteristic of corrective epithelial thickness after femtosecond laser-assisted lenticule intrastromal keratoplasty (LIKE) to correct moderate-to-high hyperopia. METHODS: The prospective case series study of the LIKE procedure was performed to correct moderate-to-high hyperopia. The epithelial thickness map was generated by anterior segment optical coherence tomography (AS-OCT) in the corneal central 9-mm zone. Keratometry and corneal higher order aberrations were analyzed by Pentacam (Oculus Optikgeräte GmbH) preoperatively and postoperatively. RESULTS: In the 26 eyes of 13 participants who underwent the LIKE procedure for moderate-to-high hyperopia, the attempted spherical equivalence (SEQ) was +6.50 ± 1.09 diopters (D). Compared to the preoperative epithelial thickness maps, the postoperative epithelial thickness had become significantly thinner in the central 5-mm zone; the difference was 6 to 7 µm. The paracentral epithelium performed nonuniform remodeling; the thinnest epithelial thickness was located in the inferotemporal section, which has the greatest difference from the superonasal; the difference between these two was approximately 3 µm. Through correlation analysis, it was found that the sections with thinner epithelium were significantly related to corneal curvature and corneal vertical coma. CONCLUSIONS: The LIKE procedure can be used to correct moderate-to-high hyperopia. This study further indicated the epithelial remodeling characteristic after the LIKE procedure: the central and paracentral corneal epithelial thickness becomes thinner, and the epithelial thickness distributes non-uniformly, which may be the important factor of the postoperative curvature asymmetric distribution and induction of corneal vertical coma. [J Refract Surg. 2024;40(5):e321-e327.].

Salzmann's Nodular Degeneration in Refractive Surgery: The Earlier Hit Hypothesis of EBM Injury-Related Fibrosis of the Subepithelial Space and Deeper Corneal Extension.

PURPOSE: To review the atypical development of Salzmann's nodular degeneration (SND) after two cases of laser in situ keratomileusis (LASIK) and one case of photorefractive keratomileusis (PRK), and to highlight the pathophysiology of SND and its treatment. METHODS: Three cases of SND (two following LASIK performed with microkeratomes and one following PRK) were reviewed and Pubmed.gov and internet searches were performed. RESULTS: SND is myofibroblast-generated fibrosis in the subepithelial space between the epithelium and Bowman's layer that develops years or decades after traumatic, surgical, infectious, or inflammatory injuries to the cornea in which the epithelial basement membrane is damaged in one or more locations and does not fully regenerate. It is hypothesized based on these cases, and the previous immunohistochemistry of other investigators, that myofibroblast precursors, such as fibrocytes or corneal fibroblasts, that enter the subepithelial space are driven to develop into myofibroblasts, which slowly proliferate and extend the fibrosis, by transforming growth factor-beta from epithelium and tears that passes through the defective epithelial basement membrane. These myofibroblasts and the disordered collagens, and other extracellular matrix components they produce, make up the subepithelial opacity characteristic of SND. Nodules are larger accumulations of myofibroblasts and disordered extracellular matrix. If the injury is associated with damage to the underlying Bowman's layer and stroma, as in LASIK flap generation, then the myofibroblasts and fibrosis can extend into Bowman's layer and the underlying anterior stroma. CONCLUSIONS: SND fibrosis often extends into Bowman's layer and the anterior stroma if there are associated Bowman's defects, such as incisions or lacerations. In the latter cases, SND frequently cannot be removed by simple scrape and peel, as typically performed for most common SND cases, but can be trimmed to remove the offending tissue. This condition is more accurately termed Salzmann's subepithelial fibrosis. [J Refract Surg. 2024;40(5):e279-e290.].

The effect of sodium hyaluronate on dry eye and corneal epithelial thickness following cataract surgery.

PURPOSE: To evaluate the effects of sodium hyaluronate drops on dry eye parameters and corneal epithelial thickness following cataract surgery. METHODS: The study included 84 patients who underwent uncomplicated phacoemulsification. In Group A, 0.15% sodium hyaluronate drops were added to the postoperative antibiotic/anti-inflammatory treatment. In Group B, only antibiotic/anti-inflammatory treatment was applied. Preoperatively and at 1 week and 1 month postoperatively, all the patients were evaluated in respect of tear break-up time (TBUT), the Schirmer test under anesthesia, the corneal fluorescein staining (CFS) score, mean central corneal thickness (CCT) and mean central corneal epithelial thickness (CCET), and the two groups were compared. RESULTS: A statistically significant difference was determined between the two groups at postoperative 1 month in respect of TBUT, Schirmer test, CFS score, and CCET (p < 0.01). In Group A, a statistically significant increase was determined in the TBUT and Schirmer values at 1 month postoperatively (p < 0.01, p = 0.01, respectively) and in Group B, these values were decreased compared to preoperatively (p < 0.01). The CCET was determined to be significantly thinner in Group B 1 month postoperatively (p < 0.01). A significant increase in CCT was observed in both groups at postoperative 1 week (p < 0.01) and preoperative values were reached at 1 month postoperatively. CONCLUSION: In the patient group using sodium hyaluronate, significant differences were determined in all dry eye parameters and CCET. The use of hyaluronate sodium drops after cataract surgery was seen to improve dry eye parameters and contribute to a healthy ocular surface by ensuring continuity of the corneal epithelium.

Age-Related Differences in the Mouse Corneal Epithelial Transcriptome and Their Impact on Corneal Wound Healing.

Purpose: Aging is a risk factor for dry eye. We sought to identify changes in the aged mouse corneal epithelial transcriptome and determine how age affects corneal sensitivity, re-epithelialization, and barrier reformation after corneal debridement. Methods: Corneal epithelium of female C57BL/6J (B6) mice of different ages (2, 12, 18, and 24 months) was collected, RNA extracted, and bulk RNA sequencing performed. Cornea sensitivity was measured with an esthesiometer in 2- to 3-month-old, 12- to 13-month-old, 18- to 19-month-old, and 22- to 25-month-old female and male mice. The 2-month-old and 18-month-old female and male mice underwent unilateral corneal debridement using a blunt blade. Wound size and fluorescein staining were visualized and photographed at different time points, and a re-epithelialization rate curve was calculated. Results: There were 157 differentially expressed genes in aged mice compared with young mice. Several pathways downregulated with age control cell migration, proteoglycan synthesis, and collagen trimerization, assembly, biosynthesis, and degradation. Male mice had decreased corneal sensitivity compared with female mice at 12 and 24 months of age. Aged mice, irrespective of sex, had delayed corneal re-epithelialization in the first 48 hours and worse corneal fluorescein staining intensity at day 14 than young mice. Conclusions: Aged corneal epithelium has an altered transcriptome. Aged mice regardless of sex heal more slowly and displayed more signs of corneal epithelial defects after wounding than young mice. These results indicate that aging significantly alters the corneal epithelium and its ability to coordinate healing.

Mucoadhesive phenylboronic acid-grafted carboxymethyl cellulose hydrogels containing glutathione for treatment of corneal epithelial cells exposed to benzalkonium chloride.

Benzalkonium chloride (BAK) is the most commonly-used preservative in topical ophthalmic medications that may cause ocular surface inflammation associated with oxidative stress and dry eye syndrome. Glutathione (GSH) is an antioxidant in human tears and able to decrease the proinflammatory cytokine release from cells and reactive oxygen species (ROS) formation. Carboxymethyl cellulose (CMC), a hydrophilic polymer, is one of most commonly used artificial tears and can promote the corneal epithelial cell adhesion, migration and re-epithelialization. However, most of commercial artificial tears provide only temporary relief of irritation symptoms and show the short-term treatment effects. In the study, 3-aminophenylboronic acid was grafted to CMC for increase of mucoadhesive properties that might increase the precorneal retention time and maintain the effective therapeutic concentration on the ocular surface. CMC was modified with different degree of substitution (DS) and characterized by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Phenylboronic acid (PBA)-grafted CMC hydrogels have interconnected porous structure and shear thinning behavior. Modification of CMC with high DS (H-PBA-CMC) shows the strong bioadhesive force. The optimal concentration of GSH to treat corneal epithelial cells (CECs) was evaluated by cell viability assay. H-PBA-CMC hydrogels could sustained release GSH and decrease the ROS level. H-PBA-CMC hydrogels containing GSH shows the therapeutic effects in BAK-damaged CECs via improvement of inflammation, apoptosis and cell viability. After topical administration of developed hydrogels, there was no ocular irritation in rabbits. These results suggested that PBA-grafted CMC hydrogels containing GSH might have potential applications for treatment of dry eye disease.

[Severe Conjunctival and Corneal Epithelial Dysplasia: A Case Series]. / Schwere konjunktivale und korneale epitheliale Dysplasie: eine Fallserie.

BACKGROUND/OBJECTIVES: Ocular surface squamous neoplasia (OSSN) are among the most frequent non-pigmented malignancies of the ocular surface. They have a wide range of histological characteristics - ranging from mild epithelial dysplasia to invasive carcinoma of the squamous cells of the cornea. They may be restricted to the conjunctiva or also involve the cornea. As there are no leading symptoms in the early stages, diagnosis may be very delayed in patients who do not receive regular ophthalmological treatment. The present case series describes clinical and histological data on OSSN and includes clinical and histological data on OSSN, including possible clinical presentations, important risk factors, special histological and cytological features and therapeutic options. METHODS: Retrospective case series of patients with histologically confirmed severe epithelial dysplasia of the conjunctiva and cornea consistent with OSSN who presented to the Department of Ophthalmology in Basel University Hospital. The analysis covered demographic data, symptoms, diagnostic testing (photo documentation, brush biopsy), treatment and cytological and/or histological material and findings. RESULTS: We report on five patients aged between 41 and 92 years at the time of diagnosis. The histological findings in all patients included severe epithelial dysplasia, but with a heterogenous clinical presentation. In all cases, the lesion started in the conjunctiva, but traversed the limbus and extended to the cornea. The primary treatment was always surgical removal. In one patient, this had to be repeated several times due to recurrent metaplasia and was complemented by subsequent mitomycin C therapy. The clinical outcome ranged between total restitution of the original state to inevitable enucleation. CONCLUSION: The clinical presentation of OSSN is highly heterogenous, so that the initial diagnosis is difficult. There are no official guidelines for treatment, so that the treatment of choice varied between clinics. Regular ophthalmological follow-ups are recommended, even after complete surgical excision. Possible relevant concomitant diseases and risk factors must be identified before therapy.

Anti-inflammatory effect of curcuminoids and their analogs in hyperosmotic human corneal limbus epithelial cells.

BACKGROUND: To assess the efficacy of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin [BDC]) and their analogs (tetrahydrocurcumin [THC], tetrahydrodemethoxycurcumin [THDC], tetrahydrobisdemethoxycurcumin) in reducing inflammatory cytokines and their toxicity to primary human corneal limbal epithelial cells, these cells were cultured and exposed to these compounds. METHODS: The PrestoBlue assay assessed cell viability after treatment. Anti-inflammatory effects on hyperosmotic cells were determined using real-time polymerase chain reaction and significance was gauged using one-way analysis of variance and Tukey's tests, considering p-values < 0.05 as significant. RESULTS: Curcuminoids and their analogs, at 1, 10, and 100 µM, exhibited no effect on cell viability compared to controls. However, cyclosporin A 1:500 significantly reduced cell viability more than most curcuminoid treatments, except 100 µM curcumin and BDC. All tested curcuminoids and analogs at these concentrations significantly decreased mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17 A, matrix metallopeptidase-9, and intercellular adhesion molecule-1 after 90 mM NaCl stimulation compared to untreated cells. Furthermore, proinflammatory cytokine levels from hyperosmotic cells treated with 1, 10, and 100 µM curcumin, 100 µM BDC, 100 µM THC, 1 and 100 µM THDC mirrored those treated with cyclosporin A 1:500. CONCLUSION: The anti-inflammatory efficiency of 1 and 10 µM curcumin, 100 µM THC, 1 and 100 µM THDC was comparable to that of cyclosporin A 1:500 while maintaining cell viability.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells.

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.

Clinical features and comprehensive treatment of persistent corneal epithelial dysfunction after cataract surgery.

OBJECTIVE: Evaluation of clinical efficacy and safety of tobramycin/dexamethasone eye ointment in treating persistent corneal epithelial dysfunction (PED) after cataract surgery. METHODS: 26 cases diagnosed as PED after cataract surgery accept the tobramycin/dexamethasone ophthalmic ointment and intense pulse light treatment in the Xiamen University of Xiamen eye center between September 2016 and April 2022 were retrospectively analyzed, mainly including clinical manifestations, characteristics of morphological changes imaged by in vivo confocal microscopy, meibomian glands infrared photography, lipid layer thickness (LLT), management and therapeutic effects. RESULTS: There were 26 eyes, include 8(35%) males and 15(65%) females with an average age of 69.6 ± 5.2 years(50 to 78 years). The mean hospitalization time was (18.4 ± 7.5) days after cataract surgery. Twenty patients had meibomian gland dysfunction. Infrared photography revealed varying loss in the meibomian glands, with a mean score of 3.8 ± 1.2 for gland loss. The mean LLT was 61.6 ± 8.4 nm. After treatment, 20 patients were cured, and 3 received amniotic membrane transplantation. After treatment, the uncorrected visual acuity (UCVA) and best-corrected vision activity (BCVA) improved (P < 0.001), and there was no significant difference in intraocular pressure (IOP) before and after treatment (P > 0.05). CONCLUSIONS: The early manifestation of PED after surgery is punctate staining of the corneal epithelium. Tobramycin and dexamethasone eye ointment bandages have a good repair effect. The meibomian gland massage combined with intense pulse light treatment can effectively shorten the course of the disease.

Organophosphorus Flame Retardant TPP-Induced Human Corneal Epithelial Cell Apoptosis through Caspase-Dependent Mitochondrial Pathway.

A widely used organophosphate flame retardant (OPFR), triphenyl phosphate (TPP), is frequently detected in various environmental media and humans. However, there is little known on the human corneal epithelium of health risk when exposed to TPP. In this study, human normal corneal epithelial cells (HCECs) were used to investigate the cell viability, morphology, apoptosis, and mitochondrial membrane potential after they were exposed to TPP, as well as their underlying molecular mechanisms. We found that TPP decreased cell viability in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of 220 µM. Furthermore, TPP significantly induced HCEC apoptosis, decreased mitochondrial membrane potential in a dose-dependent manner, and changed the mRNA levels of the apoptosis biomarker genes (Cyt c, Caspase-9, Caspase-3, Bcl-2, and Bax). The results showed that TPP induced cytotoxicity in HCECs, eventually leading to apoptosis and changes in mitochondrial membrane potential. In addition, the caspase-dependent mitochondrial pathways may be involved in TPP-induced HCEC apoptosis. This study provides a reference for the human corneal toxicity of TPP, indicating that the risks of OPFR to human health cannot be ignored.

The new preservative-free ophthalmic formulation of bilastine 0.6% preserves the ocular surface epithelial integrity in a comparative in vitro study.

Allergic conjunctivitis (AC) is the most common form of allergic eye disease and an increasingly prevalent condition. Topical eye drop treatments are the usual approach for managing AC, although their impact on the ocular surface is not frequently investigated. The aim of this study was to perform a comparative physicochemical characterization, and in vitro biological evaluations in primary conjunctival and corneal epithelial cells of the new multidose preservative-free bilastine 0.6% and main commercially available eye drops. MTT assay was used to measure cell viability; oxidative stress was analyzed with a ROS-sensitive probe; and apoptosis was evaluated monitoring caspase 3/7 activation. Differences in pH value, osmolarity, viscosity and phosphate levels were identified. Among all formulations, bilastine exhibited pH, osmolarity and viscosity values closer to tear film (7.4, 300 mOsm/l and ~ 1.5-10 mPa·s, respectively), and was the only phosphates-free solution. Single-dose ketotifen did not induce ROS production, and single-dose azelastine and bilastine only induced a mild increase. Bilastine and single-dose ketotifen and azelastine showed high survival rates attributable to the absence of preservative in its formulation, not inducing caspase-3/7-mediated apoptosis after 24 h. Our findings support the use of the new bilastine 0.6% for treating patients with AC to preserve and maintain the integrity of the ocular surface.

Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release.

INTRODUCTION: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds. METHODS: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1ß, ß3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury. RESULTS: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining. CONCLUSIONS: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Mitochondrial DNA-Activated cGAS-STING Signaling in Environmental Dry Eye.

Purpose: The cGAS-STING pathway has been shown to be an important mediator of inflammation. There is emerging evidence of the importance of this signaling cascade in a variety of inflammatory diseases settings. Here, we present evidence that the mitochondrial DNA (mtDNA) damage-mediated cGAS-STING pathway plays an important role in the induction of inflammation in environmental dry eye (DE). Methods: RT-qPCR and Western blot were used to assess the induction of the cGAS-STING pathway and inflammatory cytokines in environmental DE mouse model, primary human corneal epithelial cells (pHCECs), and patients with DE. RNA sequencing was used to determine mRNA expression patterns of high osmotic pressure (HOP)-stimulated pHCECs. mtDNA was detected with electron microscopy, flow cytometry, and immunofluorescent staining. mtDNA was isolated and transfected into pHCECs for evaluating the activation of the cGAS-STING pathway. Results: The expression levels of cGAS, STING, TBK1, IRF3, and IFNß were significantly increased in an environmental DE model and HOP-stimulated pHCECs. The STING inhibitor decreased the expression of inflammatory factors in DE. An upregulation of STING-mediated immune responses and IRF3 expression mediated by TBK1 were observed in the HOP group. HOP stimulation induced mitochondrial oxidative damage and the leakage of mtDNA into the cytoplasm. Then, mtDNA activated the cGAS-STING pathway and induced intracytoplasmic STING translocated to the Golgi apparatus. Finally, we also found activated cGAS-STING signaling in the human conjunctival blot cell of patients with DE. Conclusions: Our findings suggest that the cGAS-STING pathway is activated by recognizing cytoplasmic mtDNA leading to STING translocation, further exacerbating the development of inflammation in environmental DE.

Single nuclei transcriptomics of the in situ human limbal stem cell niche.

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGß4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGß4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.