IC3D Classification of Corneal Dystrophies-Edition 3.

PURPOSE: The International Committee for the Classification of Corneal Dystrophies (IC3D) was created in 2005 to develop a new classification system integrating current information on phenotype, histopathology, and genetic analysis. This update is the third edition of the IC3D nomenclature. METHODS: Peer-reviewed publications from 2014 to 2023 were evaluated. The new information was used to update the anatomic classification and each of the 22 standardized templates including the level of evidence for being a corneal dystrophy [from category 1 (most evidence) to category 4 (least evidence)]. RESULTS: Epithelial recurrent erosion dystrophies now include epithelial recurrent erosion dystrophy, category 1 ( COL17A1 mutations, chromosome 10). Signs and symptoms are similar to Franceschetti corneal dystrophy, dystrophia Smolandiensis, and dystrophia Helsinglandica, category 4. Lisch epithelial corneal dystrophy, previously reported as X-linked, has been discovered to be autosomal dominant ( MCOLN1 mutations, chromosome 19). Classic lattice corneal dystrophy (LCD) results from TGFBI R124C mutation. The LCD variant group has over 80 dystrophies with non-R124C TGFBI mutations, amyloid deposition, and often similar phenotypes to classic LCD. We propose a new nomenclature for specific LCD pathogenic variants by appending the mutation using 1-letter amino acid abbreviations to LCD. Pre-Descemet corneal dystrophies include category 1, autosomal dominant, punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD) ( PRDX3 mutations, chromosome 10). Typically asymptomatic, it can be distinguished phenotypically from pre-Descemet corneal dystrophy, category 4. We include a corneal dystrophy management table. CONCLUSIONS: The IC3D third edition provides a current summary of corneal dystrophy information. The article is available online at https://corneasociety.org/publications/ic3d .

Krupple-like factor 4 (KLF4) methylation signature in host cell in active viral keratitis with epithelial manifestation.

HSV1 presents as epithelial or stromal keratitis or keratouveitis and can lead to sight-threatening complications. KLF4, a critical transcription factor, and regulator of cell growth and differentiation, is essential in corneal epithelium stratification and homeostasis. Here, we want to understand the epigenetic modification specifically the methylation status of KLF4 in epithelium samples of HSV1 keratitis patients. After obtaining consent, epithelial scrapes were collected from 7 patients with clinically diagnosed HSV1 keratitis and 7 control samples (patients undergoing photorefractive keratectomy). Genomic DNA was isolated from the collected samples using the Qiagen DNeasy Kit. Subsequently, bisulfite modification was performed. The bisulphite-modified DNA was then subjected to PCR amplification using specific primers designed to target the KLF4, ACTB gene region, allowing for the amplification of methylated and unmethylated DNA sequences. The amplified DNA products were separated and visualized on a 3% agarose gel. KLF4 hypermethylation was found in 6 out of 7 (85.71%) eyes with viral keratitis, while 1 eye showed hypomethylation compared to PRK samples. Out of these 6, there were 2 each of epithelial dendritic keratitis, epithelial geographical keratitis, and neurotrophic keratitis. The patient with hypomethylated KLF4 had a recurrent case of HSV1 keratitis with multiple dendrites and associated vesicular lesions of the lip along with a history of fever. KLF4 hypermethylation in most viral keratitis cases indicated the under functioning of KLF4 and could indicate a potential association between KLF4 hypermethylation and the development or progression of HSV1 keratitis.

Corneal Epithelial Development and the Role of Induced Pluripotent Stem Cells for Regeneration.

Severe corneal disorders due to infective aetiologies, trauma, chemical injuries, and chronic cicatricial inflammations, are among vision-threatening pathologies leading to permanent corneal scarring. The whole cornea or lamellar corneal transplantation is often used as a last resort to restore vision. However, limited autologous tissue sources and potential adverse post-allotransplantation sequalae urge the need for more robust and strategic alternatives. Contemporary management using cultivated corneal epithelial transplantation has paved the way for utilizing stem cells as a regenerative potential. Humaninduced pluripotent stem cells (hiPSCs) can generate ectodermal progenitors and potentially be used for ocular surface regeneration. This review summarizes the process of corneal morphogenesis and the signaling pathways underlying the development of corneal epithelium, which is key to translating the maturation and differentiation process of hiPSCs in vitro. The current state of knowledge and methodology for driving efficient corneal epithelial cell differentiation from pluripotent stem cells are highlighted.

Comparative evaluation of corneal and limbal epithelial thickness in brachycephalic dogs with and without corneal diseases using spectral domain optical coherence tomography.

OBJECTIVE: To evaluate alterations in epithelial thickness during corneal degeneration, corneal pigmentation, and additional features observed through spectral-domain optical coherence tomography (SD-OCT) in brachycephalic dogs. ANIMALS AND PROCEDURES: The study used 55 eyes from 49 brachycephalic dogs that underwent OCT-containing ophthalmic examinations. The examined eyes were classified into corneal degeneration, corneal pigmentation, and normal groups according to corneal lesions. For each eye, corneal epithelial thickness (CET) in the central cornea and maximum limbal epithelial thickness (maxLET) in 4 quadrants of limbus (superior, inferior, nasal, and temporal) were measured from OCT images. Additional abnormal findings on OCT images, including irregular epithelium, subepithelial hyperreflectivity, and conjunctivochalasis, were also recorded. RESULTS: The corneal degeneration group had significantly thinner nasal and temporal maxLETs than that of the normal group (p < .001). In the central corneal OCT image of the corneal degeneration group, an irregular epithelium was observed in 70.6% and subepithelial hyperreflectivity in 82.4%, both of which were significantly higher than the normal group (p < .001). In a comparative analysis, the nasal, temporal, and inferior maxLETs were significantly thinner in the corneal pigmentation group than those in the normal group (p < .001, p < .001, and p = .01, respectively). CONCLUSIONS: Morphological changes in the limbal epithelium were observed in dogs with corneal degeneration and corneal pigmentation. LET reduction could be associated with their pathogenesis and would be valuable as an additional parameter for corneal diseases.

Collagen type XII is undetectable in keratoconus Bowman's layer.

PURPOSE: Corneal biomechanical failure is the hallmark of keratoconus (KC); however, the cause of this failure remains elusive. Collagen type XII (COL12A1), which localises to Bowman's layer (BL), is thought to function in stress-bearing areas, such as BL. Given the putative protective role of COL12A1 in biomechanical stability, this study aims to characterise COL12A1 expression in all corneal layers involved in KC. METHODS: TaqMan quantitative PCR was performed on 31 corneal epithelium samples of progressive KC and myopic control eyes. Tissue microarrays were constructed using full-thickness corneas from 61 KC cases during keratoplasty and 18 non-KC autopsy eyes and stained with an antibody specific to COL12A1. Additionally, COL12A1 was knocked out in vitro in immortalised HEK293 cells. RESULTS: COL12A1 expression was reduced at transcript levels in KC epithelium compared with controls (ratio: 0.58, p<0.03). Immunohistochemical studies demonstrated that COL12A1 protein expression in BL was undetectable, with reduced expression in KC epithelium, basement membrane and stroma. CONCLUSIONS: The apparent absence of COL12A1 in KC BL, together with the functional importance that COL12A1 is thought to have in stress bearing areas, suggests that COL12A1 may play a role in the pathogenesis of KC. Further studies are necessary to investigate the mechanisms that lead to COL12A1 dysregulation in KC.

Evaluation of aqueous-deficient and evaporative dry eye cases with confocal microscopy.

INTRODUCTION: Dry eye disease (DED) is an important health problem affecting hundreds of millions of people worldwide. In vivo confocal microscopy (IVCM) is a non-invasive imaging tool that can visualize ocular surface diseases. In this study, we aimed to evaluate corneal structures and inflammatory cells with IVCM in DED patients. MATERIAL AND METHODS: The patients were divided into three subgroups: group 1, consisting of 22 patients with aqueous tear insufficiency; group 2, consisting of 21 patients with evaporative type DED; and group 3, consisting of 20 healthy patients. Imaging was performed with IVCM. The corneal epithelium, nerves, stroma, endothelial and inflammatory cells were compared between groups. RESULTS: There was a significant decrease in corneal epithelial cell density in cases with DED, and there was a significant increase in corneal basal epithelial cell density in the aqueous-deficient type. Keratocyte density was significantly increased in the aqueous-deficient type. A significant decrease in the number and density of sub-basal nerves was found in aqueous-deficient cases, and an increase was found in neural pilling and folding. Dendritic cell density, size, number and area were significantly increased in the aqueous-deficient type. There was also decreased corneal endothelial cell density in DED. CONCLUSION: We evaluated pathological changes in DED on the corneal surface by IVCM. This methodology is valuable in terms of objectively evaluating how the corneal surface is affected in accordance with disease severity and in predicting poor response to treatment.

Inflammatory profile of keratoconic corneal epithelium.

BACKGROUND: Recent studies have presented inflammatory features on keratoconus (KC) and many inflammatory markers are described in the tears of patients with this disease. The KC pathogenesis is still unknown just like the correlation with inflammatory patterns. However, environmental and genetic issues may be part of the progress of KC. In addition, some systemic features, such as allergy and obesity, seem to be related to the progression of KC. Our purpose was to evaluate the neuropeptides vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), chemokines ligand 2 (CCL-2) and 5 (CCL-5), and interleukins 6 (IL-6) and 8 (IL-8) on corneal epithelial cells and blood of patients with KC and in healthy controls. In addition, the neutrophil-to-lymphocyte ratio (NLR) was evaluated to predict inflammation. METHODS: This including prospective observational study included 32 KC patients who underwent corneal crosslinking (CXL) and 32 control patients who underwent photorefractive keratectomy (PRK). Patients' corneal epithelial cells were removed surgically, and blood (buffy coat) was analyzed. Samples in triplicate were evaluated on rt-PCR for neuropeptides (VIP e NPY), interleukins (IL-6 e IL-8), and chemokines (CCL-2 and CCL-5). RESULTS: Our study showed statistically higher CCL-5 and IL-8 on corneal epithelial cells in patients with KC. Blood cells were statistically higher in VIP and NPY in the KC group. Interleukin-8 on blood cells was statistically significant in KC'S group; for CCL-2 and CCL-5 they were statistically lower in patients with KC compared with controls. NLR showed no difference between the groups. CONCLUSIONS: Our data support the findings of other studies that suggested altering KC status, such as inflammatory corneal disease. The presence of IL-8 in the cornea and blood samples of KC's group suggested systemic disease with a possible local or repercussion action. Further studies are warranted to elucidate KC pathogenesis and its correlation to systemic disease.

Suppression of NLRP3/Caspase-1/GSDMD Mediated Corneal Epithelium Pyroptosis Using Melatonin-Loaded Liposomes to Inhibit Benzalkonium Chloride-Induced Dry Eye Disease.

Introduction: Benzalkonium chloride (BAC) is widely employed as a preservative in eye drops, which will cause the death of corneal epithelial cells due to ROS production, DNA strand breakage, and mitochondrial dysfunction, resulting in dry eye disease (DED)-like changes in ocular surface tissues. In this study, Melatonin (MT) liposomes (TAT-MT-LIPs) designed by loading MT into TAT-modified liposomes have been developed, characterized, and used for inhibiting BAC-induced DED (BAC-DED). Methods: The TAT was chemically grafted onto the Mal-PEG2000-DSPE by Michael's addition between the sulfhydryl group in TAT and the maleimide group in Mal-PEG2000-DSPE. TAT-MT-LIPs were prepared using film dispersion followed by the extrusion method and topically treated in rats once a day. BAC-DED was induced in rats by topical administration with 0.2% BAC twice daily. Defects, edema, and inflammation of the corneas, as well as IOP, were examined. Histologic analyses of corneas were performed to assess the change of mitochondrial DNA oxidation and NLRP3/Caspase-1/GSDMD signaling transduction. Results: After topical administration, TAT-MT-LIPs significantly alleviated DED-clinical symptoms of experimental animals by inhibiting tissue inflammation and preventing the loss of the corneal epithelium and conjunctival goblet cells. Our data suggested continuous ocular surface exposure of BAC-induced NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis, which was not reported before. BAC caused substantial mt-DNA oxidation, which promoted the transduction of NLRP3/Caspase-1/GSDMD and consequent corneal epithelium pyroptosis. TAT-MT-LIPs could efficiently suppress the BAC-induced corneal epithelium pyroptosis and inflammation by inhibiting mt-DNA oxidation and the subsequent signal transmission. Conclusion: NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis is involved in the development of BAC-DED. The present study provided new insights into the adverse effects of BAC, which can serve as a new target for protecting corneal epithelium when applying BAC as a preservative in eye drops. The developed TAT-MT-LIPs can efficiently inhibit BAC-DED and give great potential to be developed as a new DED treatment.

Role of in vivo confocal microscopy in dry eye disease and eye pain.

Dry eye disease is known to have a lot of variability in presentation with overlapping subtypes. Understanding the pathology of this condition will guide therapeutic options. In vivo confocal microscopy is a diagnostic and imaging modality that provides high magnification and high-resolution images of all layers of the cornea and ocular surface. Various structures in the cornea and their alterations due to dry eye have been imaged. The impact of the tear film instability, inflammation, and altered homeostasis on the corneal epithelium, nerves, keratocytes, and dendritic cells have been evaluated across different studies. In addition, key features of IVCM in patients with neuropathic pain have been highlighted in this paper.

[Epithelial basement membrane corneal dystrophy: A case report]. / Distrofia de membrana basal epitelial corneal: a propósito de un caso.

Epithelial basement membrane corneal dystrophy is a rare entity, characterized by recurrent corneal erosions secondary to a disorder in the attachment of the corneal epithelium to the basement membrane. To date, mainly the ophthalmological aspect of cases has been reported, with little emphasis on the pathology of this lesion. Here we aim to describe the microscopy and discuss the clinical and therapeutic aspects of a case.

Pre-Clinical Evaluation of Efficacy and Safety of Human Limbus-Derived Stromal/Mesenchymal Stem Cells with and without Alginate Encapsulation for Future Clinical Applications.

Corneal opacification or scarring is one of the leading causes of blindness worldwide. Human limbus-derived stromal/mesenchymal stem cells (hLMSCs) have the potential of clearing corneal scarring. In the current preclinical studies, we aimed to determine their ability to heal the scarred corneas, in a murine model of corneal scar, and examined their ocular and systemic toxicity after topical administration to rabbit eyes. The hLMSCs were derived from human donor corneas and were cultivated in a clean room facility in compliance with the current good manufacturing practices (cGMP). Before the administration, the hLMSCs were analyzed for their characteristic properties including immunostaining, and were further subjected to sterility and stability analysis. The corneas (right eye) of C57BL/6 mice (n = 56) were stripped of their central epithelium and superficial anterior stroma using a rotary burr (Alger Brush® II). Few mice were left untreated (n = 8), while few (n = 24) were treated immediately with hLMSCs after debridement (prophylaxis group). The rest (n = 24, scar group) were allowed to develop corneal scarring for 2 weeks and then treated with hLMSCs. In both groups, the treatment modalities included encapsulated (En+) and non-encapsulated (En-) hLMSCs and sham (vehicle) treatment. The follow-up (4 weeks) after the treatment or debridement included clinical photography, fluorescein staining, and optical coherence tomography at regular intervals. All the images and scans were analyzed using ImageJ software to assess the changes in corneal haze, scar area, and the reflectivity ratio of the epithelium to the stroma. The scar area and the scar intensity were found to be decreased in the groups that received hLMSCs. The reflectivity of the stroma was found to be normalized to the baseline levels before the debridement in the eyes that were treated with hLMSCs, relative to the untreated. In the safety study, the central corneas of the left eye of 18 New Zealand rabbits were scraped with a needle and then treated with En+ hLMSCs, En- hLMSCs, and the sham (n = 6 each). Rabbits were then followed up for 4 weeks, during which blood and tear samples were collected at regular intervals. These rabbits were then assessed for changes in the quantities of inflammatory markers (TNF-α, IL-6, and IgE) in the sera and tears, changes in the ocular surface observations such as intraocular pressure (IOP), and the hematological and clinical chemistry parameters. Four weeks later, the rabbits were euthanized and examined histopathologically. No significant changes in conjunctival congestion, corneal clarity, or IOP were noticed during the ophthalmic examination. The level of inflammatory molecules (TNF-α and IL-6 TNF-α) and the hematological parameters were similar in all groups without any significant changes. Histological examination of the internal organs and ocular tissues did not reveal any abnormalities. The results of these studies summarize that the En+ and En- hLMSCs are not harmful to the recipient and potentially restore the transparency of debrided or scarred corneas, indicating that hLMSCs can be assessed for clinical use in humans.

Clinical Trial of Autologous Cultivated Limbal Epithelial Cell Sheet Transplantation for Patients with Limbal Stem Cell Deficiency.

PURPOSE: To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). DESIGN: A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. PARTICIPANTS: Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. METHODS: A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. MAIN OUTCOME MEASURE: Corneal epithelial reconstruction rate was the primary end point. RESULTS: Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. CONCLUSIONS: The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Novel Animal Model of Limbal Stem Cell Deficiency Induced by Forcing Eye-Open at Birth.

PURPOSE: The aim of this study was to develop a rat model of limbal stem cell deficiency (LSCD) by forcing eye-open at birth (FEOB). METHODS: A total of 200 Sprague-Dawley neonatal rats were randomly divided into the control group and the experimental group, which received eyelid open surgery on postnatal day 1 (P1). Observation time points were defined as P1, P5, P10, P15, and P30. Slit-lamp microscope and corneal confocal microscope were used to observe the clinical features of the model. The eyeballs were collected for hematoxylin and eosin staining and periodic acid-Schiff staining. Proliferating cell nuclear antigen, CD68/polymorphonuclear leukocytes, and cytokeratin 10/12/13 immunostaining were performed, while the ultrastructure of the cornea was observed by scanning electron microscopy. Real-time polymerase chain reactions (PCRs), western blot, and immunohistochemical staining of activin A receptor-like kinase-1/5 were used to analyze the possible pathogenesis. RESULTS: FEOB could successfully induce the typical manifestations of LSCD, including corneal neovascularization, severe inflammation, and corneal opacity. In the FEOB group, goblet cells could be detected in the corneal epithelium by periodic acid-Schiff staining. The expression of cytokeratins was also different between the 2 groups. Furthermore, proliferating cell nuclear antigen immunohistochemical staining revealed the weak proliferation and differentiation ability of limbal epithelial stem cells in the FEOB group. Real-time PCRs, western blot, and immunohistochemical staining of activin A receptor-like kinase-1/activin A receptor-like kinase-5 in the FEOB group showed different expression patterns than those of the control group. CONCLUSIONS: FEOB in rats induces ocular surface changes resembling LSCD in humans, representing a novel model of LSCD.

Corneal dendritic cells in diabetes mellitus: A narrative review.

Diabetes mellitus is a global public health problem with both macrovascular and microvascular complications, such as diabetic corneal neuropathy (DCN). Using in-vivo confocal microscopy, corneal nerve changes in DCN patients can be examined. Additionally, changes in the morphology and quantity of corneal dendritic cells (DCs) in diabetic corneas have also been observed. DCs are bone marrow-derived antigen-presenting cells that serve both immunological and non-immunological roles in human corneas. However, the role and pathogenesis of corneal DC in diabetic corneas have not been well understood. In this article, we provide a comprehensive review of both animal and clinical studies that report changes in DCs, including the DC density, maturation stages, as well as relationships between the corneal DCs, corneal nerves, and corneal epithelium, in diabetic corneas. We have also discussed the associations between the changes in corneal DCs and various clinical or imaging parameters, including age, corneal nerve status, and blood metabolic parameters. Such information would provide valuable insight into the development of diagnostic, preventive, and therapeutic strategies for DM-associated ocular surface complications.

Future regenerative therapies for corneal disease.

PURPOSE OF REVIEW: To highlight the progress and future direction of stem-cell based regenerative therapies for the treatment of corneal disease. RECENT FINDINGS: Corneal stem cell-based therapies, such as limbal stem cell transplantation, corneal stromal stem cell transplantation, endothelial stem cell transplantation, and stem cell-derived extracellular vesicles have demonstrated promising results in the laboratory. Although most are still in preclinical development or early phase clinical trials, these stem cell-based therapies hold potential to facilitate tissue regeneration, restore native function, and inhibit pathologic disease processes such as fibrosis, inflammation, and neovascularization. SUMMARY: Stem cell-based therapy offers a promising therapeutic option that can circumvent several of the challenges and limitations of traditional surgical treatment. This concise review summarizes the progress in stem-cell based therapies for corneal diseases along with their history, underlying mechanisms, limitations, and future areas for development.

Management of limbal stem cell deficiency by amnion-assisted conjunctival epithelial redirection using vacuum-dried amniotic membrane and fibrin glue.

PURPOSE: To study the outcome of a modified amnion-assisted conjunctival epithelial redirection (ACER) technique using vacuum-dried amnion (Omnigen) and fibrin glue for managing total limbal stem cell deficiency (LSCD). METHOD: A retrospective, interventional case series of all patients with total LSCD who underwent limbal stem cell transplant (LSCT) using the modified ACER procedure between 2016 and 2019. The outcome was defined as: (1) success: complete corneal re-epithelialisation without conjunctivalisation; (2) partial success: sub-total corneal re-epithelialisation with partial non-progressive conjunctivalisation sparing the visual axis and (3) failure: conjunctivalisation affecting the visual axis. RESULTS: Ten patients (six men), with a mean age of 46.2±18.4 years, were included. The mean follow-up was 23.0±13.9 months. Causes of LSCD were chemical eye injury (30%), congenital aniridia-related keratopathy (30%), ocular surface malignancy (20%), Steven-Johnson syndrome (10%) and contact lens overuse (10%). 50% were bilateral. The time from diagnosis to ACER (for acquired causes) was 45.6±44.4 months. 80% of patients achieved a complete/partial success following ACER and 20% of patients required repeat LSCT. Auto-LSCT was associated with a significantly higher chance of success than allo-LSCT (p=0.048). The mean best-corrected-visual-acuity (logMAR) improved significantly from 1.76±0.64 preoperatively to 0.94±0.94 at final follow-up (p=0.009). Omnigen was available off-the-shelf stored at room temperature and its transparency enabled visualisation of the healing epithelium beneath. CONCLUSION: LSCT using the modified ACER serves as an effective ocular surface reconstruction technique in managing total LSCD and improving vision. Vacuum-dried amnion provides advantages of easy handling, transparency and storage at room temperature.

Susceptibility of the Intact and Traumatized Feline Cornea to In Vitro Binding and Invasion by Acanthamoeba castellanii.

PURPOSE: Acanthamoeba castellanii ( A. castellanii ) displays host specificity at the level of the ocular surface. This study determined the susceptibility of the intact and traumatized feline cornea to A. castellanii binding and invasion relative to other host species with established susceptibility and resistance to Acanthamoeba binding. METHODS: Full-thickness buttons of fresh feline, porcine, and canine corneas were prepared. The corneal epithelium was confirmed intact by fluorescein staining or lightly scarified with a 25-G needle to simulate corneal trauma. Acanthamoeba castellanii was axenically cultivated. Corneal buttons were incubated with the parasite suspension or parasite-free medium for 18 hours at 35°C. Corneal buttons were rinsed, fixed, and processed for histopathology and immunohistochemistry using immunoperoxidase and immunofluorescence methods of amoeba detection. RESULTS: Numerous amoebae were bound to feline and porcine corneas incubated with parasites. In both intact and traumatized corneas, amoebae were detected at all levels in the corneal epithelium and within the anterior stroma. In traumatized corneal sections, amoebae were frequently present in regions of epithelial damage. Corneal architecture was well-preserved in sections incubated with parasite-free medium; however, epithelial cell sloughing, separation of epithelial layers, and epithelial detachment from the stroma were observed in corneas incubated with amoebae. Intact and traumatized canine corneas were relatively free of adherent amoebae, and corneal architecture was indistinguishable between sections incubated with the parasite suspension and parasite-free medium. CONCLUSIONS: The feline cornea is highly susceptible to in vitro binding and invasion by A. castellanii . Acanthamoeba binding to the feline cornea does not require a previous epithelial defect.

The TGM2 inhibitor cysteamine hydrochloride does not impact corneal epithelial and stromal wound healing in vitro and in vivo.

Corneal wound healing is integral for resolution of corneal disease or for post-operative healing. However, corneal scarring that may occur secondary to this process can significantly impair vision. Tissue transglutaminase 2 (TGM2) inhibition has shown promising antifibrotic effects and thus holds promise to prevent or treat corneal scarring. The commercially available ocular solution for treatment of ocular manifestations of Cystinosis, Cystaran®, contains the TGM2 inhibitor cysteamine hydrochloride (CH). The purpose of this study is to assess the safety of CH on corneal epithelial and stromal wounds, its effects on corneal wound healing, and its efficacy against corneal scarring following wounding. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were first used to quantify and localize TGM2 expression in the cornea. Subsequently, (i) the in vitro effects of CH at 0.163, 1.63, and 16.3 mM on corneal epithelial cell migration was assessed with an epithelial cell migration assay, and (ii) the in vivo effects of application of 1.63 mM CH on epithelial and stromal wounds was assessed in a rabbit model with ophthalmic examinations, inflammation scoring, color and fluorescein imaging, optical coherence tomography (OCT), and confocal biomicroscopy. Post-mortem assessment of corneal tissue post-stromal wounding included biomechanical characterization (atomic force microscopy (AFM)), histology (H&E staining), and determining incidence of myofibroblasts (immunostaining against α-SMA) in wounded corneal tissue. TGM2 expression was highest in corneal epithelial cells. Application of the TGM2 inhibitor CH did not affect in vitro epithelial cell migration at the two lower concentrations tested. At 16.3 mM, decreased cell migration was observed. In vivo application of CH at 57 mM was well tolerated and did not adversely affect wound healing. No difference in corneal scarring was found between CH treated and vehicle control eyes. This study shows that the TGM2 inhibitor CH, at the FDA-approved dose, is well tolerated in a rabbit model of corneal wound healing and does not adversely affect epithelial or stromal wound healing. This supports the safe use of this medication in Cystinosis patients with open corneal wounds. CH did not have an effect on corneal scarring in this study, suggesting that Cystaran® administration to patients with corneal wounds is unlikely to decrease corneal fibrosis.

Targeting Type I IFN/STAT1 signaling inhibited and reversed corneal squamous metaplasia in Aire-deficient mouse.

Corneal transparency and integrity are essential for obtaining good vision; nevertheless, squamous metaplasia (SQM) of ocular epithelium is a kind of serious blinding corneal diseases, without therapeutic medication in clinic. Here, we found that deficiency of the autoimmune regulator (AIRE) in corneas spontaneously developed corneal plaques. Using corneal abrasion model, we revealed that deletion of Aire not only resulted in delayed corneal re-epithelialization, but also promoted a cell-fate transition from transparent corneal epithelium to keratinized epithelium, histopathologically characterized with SQM based on the transcriptomic analysis. Mechanistically, Aire-deficient corneas led to the heightened Type I interferon (IFN-I)/STAT1 signaling after abrasion. Pharmacological blockade of IFN-I/JAK/STAT1 signaling in Aire-knockout (KO) corneas not only accelerated epithelial wound healing, but also alleviated corneal plaques and SQM. Collectively, our findings revealed critical roles of AIRE in governing corneal epithelial homeostasis and pathologic keratinization, and further identified IFN-I/STAT1 signaling as a potential target for treating ocular surface diseases with SQM, and even for treating pathological scenarios related to SQM in other tissues.

Evaluation of the Algerbrush II rotating burr as a tool for inducing ocular surface failure in a mouse model.

Purpose: The Algerbrush II has been widely used to induce corneal and limbal injuries in animal models. The extent of injury varies with the duration of exposure, pressure from the placement of the burr, and the size of the burr. However, no study has explored the correlation between the duration of exposure and the severity of injury in mouse model with corneal and limbal stem cell deficiency (LSCD) induced using the Algerbrush II. Therefore, this study aimed to evaluate the variations in the severity of corneal and limbal injury with different durations of the Algerbrush II application. Methods: The entire cornea and limbus of C57BL/6 mice were injured for 30-45 s, 60-75 s, 90-120 s, and 3-4 min. Photography and slit-lamp examination was performed on days 0, 2, 4, and 7, followed by hematoxylin & eosin, periodic acid-Schiff, and immunohistochemical staining. Statistical analysis was performed using one way ANOVA analysis. Results: A duration of 30-45 s of injury was found to be sufficient to induce superficial corneal and limbal epithelial debridement and re-epithelialization was completed in all eyes by day 7; however, clinical signs of LSCD were not observed in all mice. Increasing the exposure time to 90-120 s resulted in central 2+ corneal opacity with limbal and paracentral corneal neovascularization. All eyes injured for 3-4 min displayed clinical signs of LSCD, such as persistent epithelial defects on day 7 after the injury, central corneal neovascularization, and 2.2+ diffuse corneal opacity. Histological signs of LSCD, including goblet cell metaplasia and K13 expression on the corneal surface, were observed in all injured eyes. Conclusions: Our findings suggest that the duration of injury is an important factor influencing the severity of LSCD in a murine model of injury. A 1-mm rotating burr was found to be more effective for keratectomy and pigment release, whereas a 0.5-mm burr was more suitable for corneal epithelial debridement.