Maintaining Corneal Transparency Research Articles

The Human Cornea: Unraveling Its Structural, Chemical, and Biochemical Complexities.

The cornea, the transparent part of the anterior eye, is vital for light refraction and vision. This review examines the intricate chemical and biochemical interactions essential for maintaining corneal transparency and highlights significant advancements in corneal biology. The cornea comprises five layers: the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium, each contributing uniquely to its structure and function. The epithelium, maintained by limbal stem cells, serves as a barrier and interacts with the tear film to maintain ocular surface health. The stroma, abundant in organized collagen fibrils and regulated by proteoglycans, is crucial for corneal clarity and biomechanical integrity, whereas the endothelium regulates corneal hydration and nutrition. Recent imaging advances have improved visualization of these molecular structures, enhancing our understanding of collagen organization and cross-linking. Proteoglycans such as decorin and lumican regulate collagen spacing and hydration, directly influencing corneal clarity. Biochemical processes within the cornea involve signaling molecules, growth factors, and cytokines, which are essential for wound healing, inflammation, and injury response. Despite progress, questions remain regarding corneal wound healing mechanisms, the impact of oxidative stress, and the roles of microRNAs. This review synthesizes recent discoveries to advance our understanding of corneal physiology and biochemical functions.

Mesenchymal stem cells derived extracellular vesicles modified PLGA electrospinning nanofibrous scaffolds for corneal and retinal repair

Tissue self-renewal is crucial for ocular diseases such as corneal damage and retinal holes. In this study, a novel Poly (lactic-co-glycolic acid) (PLGA) electrospinning nanofibrous scaffold (PLGAENS), loaded with mesenchymal stem cells-derived extracellular vesicles (MSC-EVs), was developed to accelerate the healing of the cornea and retina. In-vitro experiments confirmed the supportive properties of PLGAENS, demonstrating its ability to promote cellular proliferation, migration, and extension. In the rat corneal alkali burn model and rabbit retinal hole model, MSC-EVs modified PLGAENS (PLGAMSC-EVs) accelerated the restoration of the corneal epithelium and stroma, as well as the closure of retinal holes. Additionally, miR-21-5p was identified as being enriched in MSC-EVs. Mechanistically, miR-21-5p suppressed scar formation by targeting the programmed cell death protein 4 (PDCD4) gene, reducing fibrosis and the expression of collagen-related genes, which helped maintain corneal transparency and retinal integrity. Overall, these findings underscored the potential of PLGAMSC-EVs in promoting ocular wound healing and suggested a promising new therapeutic strategy for the clinical treatment of corneal damage and retinal holes.

COL8A2 activation enhances function of corneal endothelial cells through HIPPO signaling/mitochondria pathway

Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency.

Influence of Intraocular Pressure on the Expression and Activity of Sodium-Potassium Pumps in the Corneal Endothelium.

The corneal endothelium is responsible for pumping fluid out of the stroma in order to maintain corneal transparency, which depends in part on the expression and activity of sodium-potassium pumps. In this study, we evaluated how physiologic pressure and flow influence transcription, protein expression, and activity of Na+/K+-ATPase. Native and engineered corneal endothelia were cultured in a bioreactor in the presence of pressure and flow (hydrodynamic culture condition) or in a Petri dish (static culture condition). Transcription of ATP1A1 was assessed using qPCR, the expression of the α1 subunit of Na+/K+-ATPase was measured using Western blots and ELISA assays, and Na+/K+-ATPase activity was evaluated using an ATPase assay in the presence of ouabain. Results show that physiologic pressure and flow increase the transcription and the protein expression of Na+/K+-ATPase α1 in engineered corneal endothelia, while they remain stable in native corneal endothelia. Interestingly, the activity of Na+/K+-ATPase was increased in the presence of physiologic pressure and flow in both native and engineered corneal endothelia. These findings highlight the role of the in vivo environment on the functionality of the corneal endothelium.

Advances in Cell Transplantation Therapy for Limbal Stem Cell Deficiency.

Limbal stem cells (LSCs) are essential for maintaining corneal transparency and ocular surface integrity. Many external factors or genetic diseases can lead to corneal limbal stem cell deficiency (LSCD), resulting in the loss of barrier and corneal epithelial cell renewal functions. Stem cell transplantation is one of the primary treatments for LSCD, including limbal transplantation and cultivated limbal epithelial transplantation. In addition, a variety of non-limbal stem cell lines have been experimented with for LSCD treatment. Biological scaffolds are also used to support in vitro stem cell culture and transplantation. Here, we review the mechanisms of corneal maintenance by LSCs, the clinical stage and surgical treatment of LSCD, the source of stem cells, and the biological scaffolds required for in vitro culture. This study is a narrative retrospective study aimed at collecting available information on various aspects of surgical treatments for LSCD. Relevant literature was searched in a range of online databases, including Web of Science, Scopus, and PubMed from 2005 to March, 2023. A total of 397 relevant articles were found, and 49 articles with strong relevance to the studies in this paper were obtained and analyzed. Moreover, 11 of these articles were on the concept of LSCD and the mechanism of LESCs maintaining the corneal epithelium, 3 articles on the staging and grading of LSCD, 17 articles on cell transplantation methods and donor cell sources, and 18 articles on scaffolds for delivering stem cells. We also summarized the advantages and disadvantages of different cell transplantation methods and the benefits and limitations of scaffolds based on the above literature. The treatment of LSCD is determined by the clinical stage and whether it involves monocular or binocular eyes. Appropriate surgical techniques should be taken for LSCD patients in order to reconstruct the ocular surface, relieve symptoms, and restore visual function. Meanwhile, biological scaffolds assist in the ex vivo culture and implantation of stem cells.

Central Corneal Thickness and Endothelial Cell Changes in Diabetics and Age-Matched Non-diabetics in a Tertiary Care Hospital in Central India.

Background Diabetes has become an epidemic, significantly impacting ocular health as one of its end-organ responses. Among the various ocular complications, alterations in corneal morphology stand out. Central corneal thickness (CCT) and endothelial cell function are vital parameters in assessing intraocular pressure, conducting pre-refractive surgery evaluations, and maintaining corneal transparency. Understanding these changes in diabetic individuals compared to non-diabetics is crucial for managing ocular health in this population. Aim and objective This study evaluates and compares CCT and endothelial cell changes between diabetic individuals and age-matched non-diabetics. By analyzing these parameters, the study seeks to provide insights into the impact of diabetes on corneal morphology and its implications for ocular health. Methods The study recruited 124 patients from the Ophthalmology Outpatient Department (OPD) at Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi. A cross-sectional research design was employed to collect data over six months. Patients were carefully selected, and informed consent was obtained from all participants. CCT and endothelial cell parameters were assessed using specular microscopy, a non-invasive imaging technique. Statistical analysis was done using the software Statistical Package for the Social Sciences (SPSS) for inferential statistics, such as t-tests and ANOVA, and comparing parameters between diabetic and non-diabetic groups.Findings were interpreted based on both statistical significance and clinical relevance. Results In diabetic patients, the mean CCT was 547.91 µm, while it was 523.62 µm in non-diabetic individuals.The T statistic for this variable was 5.14, indicating a17 significant differencebetween the two groups. Similarly, significant differences were found between diabetics and non-diabetics for endothelial cell density, coefficient of variation, and hexagonality, as evidenced by their respective T statistics of 7.46, 5.17, and 4.91. Endothelial cell density averaged 2375 cells/mm2 in diabetics and 2666.95 cells/mm2 in non-diabetics. Additionally, the coefficient of variation was higher among people with diabetes (40.87%) compared to non-diabetics (35.09%). Hexagonality, a measure of endothelial cell shape, was lower in diabetic corneas (40.48%) than in non-diabetic corneas (46.46%). Conclusion The study observed significant differences in corneal morphology, including central thickness and endothelial cell changes, between diabetic and non-diabetic individuals. These findings underscore the impact of diabetes on ocular health and emphasize the importance of monitoring corneal parameters in diabetic patients. Understanding these changes can aid in better management and treatment strategies for ocular complications associated with diabetes.

Electrospun Nanofiber Membrane for Cultured Corneal Endothelial Cell Transplantation.

The corneal endothelium, comprising densely packed corneal endothelial cells (CECs) adhering to Descemet's membrane (DM), plays a critical role in maintaining corneal transparency by regulating water and ion movement. CECs have limited regenerative capacity within the body, and globally, there is a shortage of donor corneas to replace damaged corneal endothelia. The development of a carrier for cultured CECs may address this worldwide clinical need. In this study we successfully manufactured a gelatin nanofiber membrane (gelNF membrane) using electrospinning, followed by crosslinking with glutaraldehyde (GA). The fabricated gelNF membrane exhibited approximately 80% transparency compared with glass and maintained a thickness of 20 µm. The gelNF membrane demonstrated desirable permeability and degradability for a Descemet's membrane analog. Importantly, CECs cultured on the gelNF membrane at high densities showed no cytotoxic effects, and the expression of key CEC functional biomarkers was verified. To assess the potential of this gelNF membrane as a carrier for cultured CEC transplantation, we used it to conduct Descemet's membrane endothelial keratoplasty (DMEK) on rabbit eyes. The outcomes suggest this gelNF membrane holds promise as a suitable carrier for cultured CEC transplantation, offering advantages in terms of transparency, permeability, and sufficient mechanical properties required for successful transplantation.

Corneal Endothelial Morphological Alterations in Type 2 Diabetes: Associations with Glycemic Control, Disease Duration, and Diabetic Retinopathy Severity—A Cross-Sectional Study in Benghazi, Libya

Background The corneal endothelium is essential for maintaining corneal transparency and fluid regulation, and its dysfunction can lead to corneal edema and decreased visual acuity. Corneal specular microscopy is used to detect corneal disorders early. Aim This study aimed to evaluate morphological changes in the corneal endothelium of patients with type 2 diabetes mellitus (DM) using specular microscopy. Methods This study enrolled 50 individuals with type 2 diabetes and 50 nondiabetic individuals as control subjects. Patients with certain ocular and systemic conditions were excluded. The collected data included demographic information, medical history, recent hemoglobin A1c (HbA1c) values, visual acuity, ophthalmological examination, and diabetic retinopathy (DR) staging. The corneal endothelial morphology was evaluated using a noncontact specular microscope, which measured corneal endothelial cell density (ECD), central corneal thickness (CCT), coefficient of variation of cell size (CV), and hexagonality. The data were analyzed using SPSS software, and the results were presented as mean ± standard deviation, numbers, and percentages. An unpaired t-test was used to compare different means, and a p-value less than 0.05 was considered statistically significant. Results A majority of diabetic patients (62%) had diabetes for more than 10 years, and 58% of them had higher than 7.5% HbA1c levels. In terms of DR severity, 46% of patients had mild nonproliferative diabetic retinopathy (NPDR), 36% had moderate NPDR, and 10% had severe NPDR. There were significant differences between diabetic and nondiabetic groups in corneal ECD (2480 ± 223 cells/mm2 for DM group vs. 2652 ± 234 cells/mm2 for non-DM), hexagonality (39.6 ± 2.8% for DM group vs. 47 ± 2.1% for non-DM), CV (42 ± 2.9% for DM group vs. 35.5 ± 2.3% for non-DM), and CCT (550 ± 14.8 µm for DM group vs. 530 ± 9.6 µm for non-DM). Patients who had diabetes for more than 10 years had significantly lower ECD (2356 cells/mm2 vs. 2689 cells/mm2), lower hexagonality (39 vs. 41%), and higher CV (43 vs. 41%) and higher CCT (553.9 ± 4.6 vs. 545.5 ± 4.0) than those with less than 10 years of diabetes. As the severity of DR increased, there was a significant decrease in ECD (from 2641 ± 194 cells/mm2 for mild NPDR to 2310 ± 82 cells/mm2 for severe PDR), a decrease in hexagonality (from 40.9 ± 3.2% for mild PDR to 37.4 ± 1.1% for severe PDR), an increase in CV (from 40.1 ± 2.3% for mild PDR to 44 ± 2.9% for severe PDR), and an increase in CCT (from 543.7 ± 13.8 for mild PDR 563.8 ± 2.9 for severe PDR). Conclusion This study revealed that type 2 diabetes is associated with significant reductions in ECD and hexagonality, as well as an increase in CV and CCT. The severity of DR and the duration of diabetes were correlated with changes in these parameters. These findings underscore the need for a comprehensive evaluation of corneal health in diabetic patients.

Loss of aquaporin 5 contributes to the corneal epithelial pathogenesis via Wnt/β-catenin pathway.

AQP5 plays a crucial role in maintaining corneal transparency and the barrier function of the cornea. Here, we found that in the corneas of Aqp5-/- mice at older than 6 months, loss of AQP5 significantly increased corneal neovascularization, inflammatory cell infiltration, and corneal haze. The results of immunofluorescence staining showed that upregulation of K1, K10, and K14, and downregulation of K12 and Pax6 were detected in Aqp5-/- cornea and primary corneal epithelial cells. Loss of AQP5 aggravated wound-induced corneal neovascularization, inflammation, and haze. mRNA sequencing, western blotting, and qRT-PCR showed that Wnt2 and Wnt6 were significantly decreased in Aqp5-/- corneas and primary corneal epithelial cells, accompanied by decreased aggregation in the cytoplasm and nucleus of β-catenin. IIIC3 significantly suppressed corneal neovascularization, inflammation, haze, and maintained corneal transparent epithelial in Aqp5-/- corneas. We also found that pre-stimulated Aqp5-/- primary corneal epithelial cells with IIIC3 caused the decreased expression of K1, K10, and K14, the increased expression of K12, Pax6, and increased aggregation in the cytoplasm and nucleus of β-catenin. These findings revealed that AQP5 may regulate corneal epithelial homeostasis and function through the Wnt/β-catenin signaling pathway. Together, we uncovered a possible role of AQP5 in determining corneal epithelial cell fate and providing a potential therapeutic target for corneal epithelial dysfunction.

IPSC-Derived Corneal Endothelial Cells.

The corneal endothelium is the innermost monolayer of the cornea that maintains corneal transparency and thickness. However, adult human corneal endothelial cells (CECs) possess limited proliferative capacity, and injuries can only be repaired by migration and enlargement of resident cells. When corneal endothelial cell density is lower than the critical level (400-500 cells/mm2) due to disease or trauma, corneal endothelial dysfunction will occur and lead to corneal edema. Corneal transplantation remains the most effective clinical treatment therapy but is limited by the global shortage of healthy corneal donors. Recently, researchers have developed several alternative strategies for the treatment of corneal endothelial disease, including the transplantation of cultured human CECs and artificial corneal endothelial replacement. Early-stage results show that these strategies can effectively resolve corneal edema and restore corneal clarity and thickness, but the long-term efficacy and safety remain to be further validated. Induced pluripotent stem cells (iPSCs) represent an ideal cell source for the treatment and drug discovery of corneal endothelial diseases, which can avoid the ethical-related and immune-related problems of human embryonic stem cells (hESCs). At present, many approaches have been developed to induce the differentiation of corneal endothelial-like cells from human induced pluripotent stem cells (hiPSCs). Their safety and efficacy for the treatment of corneal endothelial dysfunction have been confirmed in rabbit and nonhuman primate animal models. Therefore, the iPSC-derived corneal endothelial cell model may provide a novel effective platform for basic and clinical research of disease modeling, drug screening, mechanistic investigation, and toxicology testing.

Specular microscopy of the corneal endothelial cells of bovines: an ex vivo study.

The endothelium is the most posterior layer of the cornea and is essential for maintaining corneal transparency. Due to variations in corneal endothelial parameters among different species, knowledge of the normal parameters for each species is crucial. To evaluate the corneal endothelium of bovines using contact specular microscopy. Twenty eyeballs from 10 male Brangus (Bos taurus) aged 24 months were evaluated. Contact specular microscopy was performed on the central corneal area. The analyzed parameters were endothelial cell density (ECD) and endothelial cell morphology. The ECD in the central area was 1,277 cells/mm2. Regarding the morphology, mainly cells with six (74.3%), five (14.7%) and seven sides (10%) were found. There were no significant differences in ECD and morphology between left and right eyes. Contact specular microscopy facilitated the analysis and measurement of corneal endothelial parameters in bovines. The data obtained will serve as a reference for the analysis of bovine corneal endothelium.

Research advances in human corneal endothelial cell regeneration

Human corneal endothelial cells (HCECs) are the prerequisite for maintaining corneal transparency, but HCECs remain arrested at the G1 phase after embryonic development and can not proliferate and regenerate. Thus, the density of HCECs decreases spontaneously with corneal development. Systemic factors, primary corneal disease, refractive factors, glaucoma, inflammation, and trauma all can cause a massive loss of HCECs, lead to corneal edema and turbidity, and ultimately induce blindness. Currently, keratoplasty is the only effective treatment, but the scarcity of donor corneas and the limitation of corneal preservation technology restrict the availability of keratoplasty. Therefore, the most appealing way to tackle the tissue shortage problem is corneal endothelial cell regeneration. In recent years, not only the endogenous regeneration of HCECs mediated by surgery, drugs and gene therapy but also the exogenous regeneration of HCECs mediated by cell therapy have made fruitful progress. Although a number of regeneration strategies have entered the clinical trial stage, the wide clinical application of corneal endothelial regeneration is still far away. This review elaborates the basic research, clinical application and limitation of current strategies of corneal endothelial cell regeneration.

Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.

Sheep Corneal Endothelium Morphology - Evaluation with Trypan Blue and Alizarin Red

Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods & Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P < 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quantitative analyses must characterise the endothelial parameters of the different species. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research. Keywords: ovine, endothelial cells, ex vivo model, vital staining, hexagonality.

A unique F-actin and junctional organization that maintains the corneal endothelial barrier

Introduction: The corneal endothelium is responsible for the correct hydration of the corneal stroma and for an adequate transport through this tissue. Corneal endothelial layer has a particular organization to maintain its curved shape and function. The corneal endothelium has low proliferative capacity and form a monolayer that is subjected to an even mechanical tension coming from the positive pressure of the aqueous humor. Preserving their barrier function under suboptimal conditions, such as corneal pathologies, age and transplantation, is essential for maintaining corneal transparency. Material and methods: We have investigated the structure and the proteins in charge of maintaining corneal endothelial barrier function by confocal microscopy and time lapse spinning disk microscopy in murine corneas ex vivo, and hepatic organoids. Results: We have characterized a novel filamentous actin network that organizes into radial structures arising from the center of the cell under the nucleus towards cell-cell junctions. This structure is also observed in spherical epithelial organoids whose cells are also exposed to positive luminal pressure. Due to the simplicity of the model, it can be easily implemented in any clinic which leads to increasing ADR and preventing CRC, but requires validation in large multicenter trials.

Notch1 signaling in keratocytes maintains corneal transparency by suppressing VEGF expression.

SummaryThe cornea fends off chemicals, dirt, and infectious particles and provides most of the eye’s focusing power. Corneal transparency is of paramount importance to normal vision, yet how it is established and maintained remains unclear. Here, we ablated Notch1 in keratocytes using Twist2-Cre mice and found that Twist2-Cre; Notch1f/f mice developed stroma expansion and neovascularization, followed by hyperproliferation and metaplasia of corneal epithelial progenitor cells and plaque formation at central cornea, leading to loss of transparency. Development of these phenotypes does not involve bacteria-caused inflammation; instead, Notch1 deletion upregulates Vegfa and Vegfc via Hif1α in keratocytes. Vascular endothelial growth factor (VEGF) receptor inhibitor axitinib prevented development of these anomalies in Twist2-Cre; Notch1f/f mice, suggesting that VEGFs secreted by keratocytes promote not only neovascularization but also proliferation and metaplasia of epithelial progenitor cells at central cornea. This study uncovers a Notch1-Hif1α-VEGF pathway in keratocytes that maintains corneal transparency and represents a potential target for treatment of related corneal disorders.

A comparative study of endothelial cell loss in small incision cataract surgery versus phacoemulsification cataract surgery by using specular microscope at tertiary care ophthalmic centre in Bengaluru, Karnataka

Most commonly performed surgical procedure in Ophthalmology is cataract extraction by MSICS and Phacoemulsification. It has always been associated with damage to endothelium of cornea, a layer which is vital for maintaining corneal transparency. The corneal endothelium regulates stromal hydration and maintains the transparency of the cornea by constantly removing the fluid out of the corneal stroma. The density of corneal endothelial cells and its integrity is an important determinant for corneal transparency. Post-operative corneal decompensation leading to reduced visual acuity can occur as a result of this surgical trauma. Hence it is necessary to determine the surgical technique safest to corneal endothelium. Specular microscopy helps to determine this corneal endothelial cell density. A hospital based longitudinal study was done from November 2017 to May 2019 in 124 patients at Minto Ophthalmic Hospital. 62 patients underwent MSICS and 62 phacoemulsification. After a written informed consent patients were evaluated with detailed history, slit lamp examination, direct and indirect ophthalmoscopy, biometry, lacrimal syringing, IOP measurement with tonometry and endothelial cell count was evaluated using non contact specular microscopy preoperatively and postoperative 1 week and 6 weeks. Statistical data analysed by unpaired – t test. The mean ECC (cells/mm) in MSICS and phacoemulsification group preoperatively was 2486.82 + 152.730 and 2433.71 + 192.692 respectively. The mean endothelial cell loss (cells/mm) was 314.61 + 64.428 and 324.31 + 30.67 at 1 week and 345.71 + 66.68 and 354.95 + 53.885 at 6 weeks postoperatively between the two groups. The endothelial cell loss was not statistically significant at 1 week (p -0.28) and 6 weeks (p-0.39) postoperatively between the two groups. There was no clinically or statistically significant difference in the endothelial cell loss between MSICS and Phacoemulsification. As MSICS is economical and less dependent on technology, it can be a safe option in developing world.

Autophagy in Extracellular Matrix and Wound Healing Modulation in the Cornea.

Autophagy is a robust cellular mechanism for disposing of harmful molecules or recycling them to cells, which also regulates physiopathological processes in cornea. Dysregulated autophagy causes inefficient clearance of unwanted proteins and cellular debris, mitochondrial disorganization, defective inflammation, organ dysfunctions, cell death, and diseases. The cornea accounts for two-thirds of the refraction of light that occurs in the eyes, but is prone to trauma/injury and infection. The extracellular matrix (ECM) is a noncellular dynamic macromolecular network in corneal tissues comprised of collagens, proteoglycans, elastin, fibronectin, laminins, hyaluronan, and glycoproteins. The ECM undergoes remodeling by matrix-degrading enzymes and maintains corneal transparency. Autophagy plays an important role in the ECM and wound healing maintenance. Delayed/dysregulated autophagy impacts the ECM and wound healing, and can lead to corneal dysfunction. Stromal wound healing involves responses from the corneal epithelium, basement membrane, keratocytes, the ECM, and many cytokines and chemokines, including transforming growth factor beta-1 and platelet-derived growth factor. Mild corneal injuries self-repair, but greater injuries lead to corneal haze/scars/fibrosis and vision loss due to disruptions in the ECM, autophagy, and normal wound healing processes. Presently, the precise role of autophagy and ECM remodeling in corneal wound healing is elusive. This review discusses recent trends in autophagy and ECM modulation in the context of corneal wound healing and homeostasis.

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway.

PurposePrevious work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism.MethodsMNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays.ResultsIt was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway.ConclusionsThis study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.

Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment.

Corneal stroma-derived mesenchymal stem cells (CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells (LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.