Layers Of Cornea Research Articles

Chlorine-Induced Toxicity on Murine Cornea: Exploring the Potential Therapeutic Role of Antioxidants.

Chlorine (Cl2) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl2 exposure. In vivo and ex vivo, after Cl2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl2-induced damage. These results highlight the effects of Cl2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants' capacity to alleviate oxidative stress and enhance the corneal healing process.

Effect of antiglaucomatous eye drops on corneal endothelium

First choice therapy for reducing the intraocular pressure in context of glaucoma remains the applications of eye drops. Longterm exposure to topical therapy can clearly have an impact on the ocular surface, but as the medications penetrate through the cornea and are present in humour aqueous, also on all other layers of cornea, including corneal endothelium. In general, none of the medications from the standard pharmacological groups influences significantly an otherwise healthy corneal endothelium in an eye with glaucoma or ocular hypertension – be it in terms of central corneal thickness, be it in terms of endothelial cell density and morphology, be it for beta blockers, carbonic anhydrase inhibitors, pilocarpine, alpha2 agonists or prostaglandin analogues. The situation changes in diseased corneas, such as in Fuchs endothelial dystrophy, where topically applied carbonic anhydrase inhibitors can lead to increase in corneal thickness. Benzalkonium chloride has been proven toxic for corneal endothelial cells in vitro. However, in the daily practice and under real life circumstances, it is from the specific point of view on the corneal endothelium still not clear whether using non‐preserved topical glaucoma medications is an absolute imperative. One separate topic here is also a group of Rho‐kinase inhibiting topical medications. Apart from new mechanisms of action for reducing the intraocular pressure, this group of medications has also a direct influence on the corneal structures – from cornea verticillata on the epithelium corneal level, to mobilizing peripheral endothelial corneal cells as a possible therapeutic path for Fuchs endothelial dystrophy. One part of the lecture will address also these issues.

Induced pluripotent stem cells as tool for cornea study in regenerative medicine

Induced Pluripotent Stem Cells technology as tool for cornea study in regenerative medicine.The ocular surface consists of an epithelial surface with specialized regions, including cornea lacrimal gland and conjunctiva. Altogether, these regions play a role in vision maintenance by transmitting light and, as first eye layer, ensure protection for the whole eye.These major roles are subject to defective developments or imbalanced maintenance, due to genetic variants or cellular degeneration, respectively, causing vision‐related problems. Intensive research is performed world‐wide to develop new therapies for such vision defects by using in vitro models, but lack of eye donations or low viability is an obstacle for such assays. At the same time, variability among donors on their genetic or epigenetic background is the main barrier for reproducibility, in addition to advanced age of donor, which is a factor limiting cell expansion or present a high number of senescent cells.A promising approach is to use stem cells to generate ocular surface cells or tissue, by mimicking the embryo development. It exists multiple sources of stem cells able to in vitro models, adult corneal cells isolated from biopsies have the advantage to resemble the organ source, but the collection has the disadvantage to be invasive. An alternative and highly valuable approach to generate organoid is from pluripotent stem cells, carrying the potential to differentiate into most cell types of the body and are subdivided into embryonic stem cells, derived from the inner cell mass of preimplantation embryos, and induced pluripotent stem cells (IPSCs) reprogrammed from adult cells.As IPSCs are one of the most promising approaches to counteract this lack of donations, several assays were performed to select the best conditions to generate cornea cells. The main goal is to characterize the developmental procedure by growth factors modulation and assess the maturation degree for the final product. The most important aspect to generate such final product is the heterogeneity of cell population during differentiation or maturation, as epithelial cornea layer has a different cell origin in comparison to stroma or endothelial cornea.Once differentiation and maturation are achieved, this in vitro model can be used for multiple downstream applications. As therapeutic approaches, organoids can be used as a living biobank or to model corneal diseases, either by using cells from affected patients or by base editing, introducing a causative mutation into the genome. Either patient‐derived or control organoids have the high potential to develop gene therapy, optogenetics, be a source of transplantable cells to replace generating tissue or screen small molecules treatments.For this topic, we will focus on the IPSC technology to derive cornea tissue and its perspective for eye development studies, clinical or preclinical study by comparing the methodology depending of the potential applications.

Depth-resolved reconstruction of anisotropic elastic moduli in a crosslinked cornea with wave-based optical coherence elastography

Ectatic changes in the cornea are typically marked by corneal thinning with increased corneal deformation and curvature, often leading to high levels of myopia and irregular astigmatism. The most common form of ectasia is keratoconus. Corneal collagen crosslinking (CXL) is commonly used to prevent or treat keratoconus. Limited penetration of CXL into the cornea may lead to a demarcation between treated and untreated regions, suggesting a two-layer structure after the treatment. Although corneal topography can be used to monitor corneal curvature changes pre- and post-surgery, it cannot predict surgical outcomes without mapping corneal elasticity. Recent studies in non-contact dynamic optical coherence elastography (OCE) demonstrated promise in quantifying changes in corneal anisotropic stiffness induced by CXL. However, depth dependent reconstruction of corneal elastic moduli following CXL remains challenging. Here, we show that the thickness of the crosslinked corneal layer can be determined from structural OCT, whereas its mechanical moduli can be reconstructed from acoustic micro-tapping (AuT) OCE using an analytical two-layer model of guided wave propagation. In addition, we discuss how the elastic moduli of partially CXL-treated cornea layers reflect the effective engineering stiffness of the entire cornea to properly quantify corneal deformation.

Possible depth-resolved reconstruction of shear moduli in the cornea following collagen crosslinking (CXL) with optical coherence tomography and elastography.

Corneal collagen crosslinking (CXL) is commonly used to prevent or treat keratoconus. Although changes in corneal stiffness induced by CXL surgery can be monitored with non-contact dynamic optical coherence elastography (OCE) by tracking mechanical wave propagation, depth dependent changes are still unclear if the cornea is not crosslinked through the whole depth. Here, phase-decorrelation measurements on optical coherence tomography (OCT) structural images are combined with acoustic micro-tapping (AµT) OCE to explore possible reconstruction of depth-dependent stiffness within crosslinked corneas in an ex vivo human cornea sample. Experimental OCT images are analyzed to define the penetration depth of CXL into the cornea. In a representative ex vivo human cornea sample, crosslinking depth varied from ∼100 µm in the periphery to ∼150 µm in the cornea center and exhibited a sharp in-depth transition between crosslinked and untreated areas. This information was used in an analytical two-layer guided wave propagation model to quantify the stiffness of the treated layer. We also discuss how the elastic moduli of partially CXL-treated cornea layers reflect the effective engineering stiffness of the entire cornea to properly quantify corneal deformation.

Removal of grass awn from the stromal layer of cornea in a dog: A case report

Removal of grass awn from the stromal layer of cornea in a dog: A case report

Diabetes Exacerbates Pseudomonas aeruginosa Keratitis in Streptozotocin-Induced and db/db Mice via Altering Programmed Cell Death Pathways.

Patients with diabetes have a higher incidence of infections, which are often more severe. This study aimed to investigate the impact of hyperglycemia on bacterial keratitis caused by Pseudomonas aeruginosa (Pa) in two mouse models of diabetes, streptozotocin-induced type 1 diabetes mellitus (T1DM) and db/db type 2 diabetes mellitus. The susceptibility of corneas to Pa was assessed by determining the inocula required to cause infectious keratitis. Dead or dying cells were identified using TUNEL staining or immunohistochemistry. Specific inhibitors were used to evaluate the role of cell death modulators in Pa keratitis. Cytokines and Treml4 expressions were analyzed using quantitative PCR, and the role of Treml4 in keratitis was determined using small interfering RNA technology. DM corneas required significantly fewer inocula to develop Pa keratitis, with T1DM corneas requiring 750 inocula and type 2 diabetes mellitus corneas requiring 2000 inocula, compared with 10,000 inocula required for normal (NL) mice. T1DM corneas had more TUNEL-positive and fewer F4/80-positive cells than NL corneas. Phospho-caspase 8 (apoptosis) and -RIPK3 (necroptosis) staining was more intense in the epithelial and stromal layers of NL and T1DM corneas, respectively. Pa keratitis was augmented by targeting caspase-8 and prevented by RIPK3 inhibition in both NL and T1DM mice. Hyperglycemia suppressed IL-17A/F and augmented IL-17C, IL-1β, IL-1Ra, and TREML4, the downregulation of which protected T1DM corneas from Pa infection by suppressing necroptosis. RIPK3 inhibition blocked Pa infection in db/+ mice and significantly decreased the severity of keratitis in db/db mice. Hyperglycemia exacerbates bacterial keratitis in B6 mice by skewing apoptosis toward necroptosis. Preventing or reversing this transition may serve as an adjunct therapy for treating microbial keratitis in patients with diabetes.

Commentary: Clinical profile, demographic distribution, and outcomes of ocular siderosis: Electronic medical record-driven big data analytics from an eye care network in India.

Commentary: Clinical profile, demographic distribution, and outcomes of ocular siderosis: Electronic medical record-driven big data analytics from an eye care network in India.

Histological Changes Indused by Monosodium Glutamate on the Growth and Development of the Offsprings Albino Mice Cornea

This research aim to study the effect of different doses of a Monosodium Glutamate (MSG) to cause histopathological changes in the offspring corneal for the ages 7, 15, 21, 30, and 60 days. The mothers, type Mus musculus mice has been orally administrated from 17th day of pregnancy by different concentrations (2, 4, and 8 g/Kg/ body weight) of MSG using stomach tube. Microscopic examination showed no changes in the cornea for a seventh day and administrated concentrations of 2 and 4 g / kg body weight, but observed degeneration of fibre stroma, necrosis and the reduction of the anterior epithelial layer of the cornea at dose of 8 g / kg. In the group of 15 days, these changes are mentioned; hyperplasia in the anterior epithelium of the peripheral cornea of 2 g, deformation in the peripheral part of the cornea and the reduction of thickness and intensify the stroma components at a concentration of 4 g either at a concentration of 8 g / kg has appeared hyperplasia in fibroblast cells in the corneal stroma and wave of the basement membrane of anterior epithelial tissue of central cornea. In a group 21days, these changes are shown; necrosis and rarefaction in stroma of central cornea while the reduction of the anterior epithelial layer of the peripheral cornea notes and wave anterior surface of the cornea in the concentration of 2 g / kg. Deformation appeared and reduction layers and irregular with dissociation in the stromal fibers in some areas in the concentration of 4 g / kg, while in the concentration of 8 g / kg show damage in the anterior part of epithelial of the central corneal tissue. In a group 30 days and concentration of 2 g / kg, these changes were observed; necrosis, swelling in some anterior epithelial cells, condensation of nuclei in endothelial cells, with Keratinized at the surface of the cornea and edema in the stroma. Bulge appeared in the stroma of peripheral cornea and hyperplasia in fiberoblast at a concentration of 4g/kg, meanwhile at a concentration of 8 g/kg show necrosis under the anterior epithelium of central cornea and dissociation of stromal fiberoblast. In the group 60 Days at the concentration of 2 g / kg show damage in the anterior epithelial tissue of the peripheral cornea and hyperplasia in some parts of the anterior epithelial tissue in the central cornea. At the concentration of 4g/kg show damage in the stromal fibers of peripheral corneal epithelium and detachment of endothelial tissue. In concentration 8 g/kg reduction appeared in the anterior epithelial tissue in the certain part of the cornea, hyperplasia in endothelial tissue, condensed layer appeared on the surface of anterior epithelial tissue.

Layer-by-Layer Investigation of Ultrastructures and Biomechanics of Human Cornea.

The cornea is an avascular, innervated, and transparent tissue composed of five layers: the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium. It is located in the outermost fraction of the eyeball and is responsible for the refraction of two-thirds of light and protection from external mechanical damage. Although several studies have been done on the cornea on the macroscopic scale, there is a lack of studies on the micro-nanoscopic scale, especially an analysis evaluating the cornea layer by layer. In this study, atomic force microscopy (AFM) was employed to assess four layers that form the cornea, analyzing: adhesion, stiffness, and roughness. The results showed microvilli in the epithelial and endothelial layers, pores in the basement membrane, and collagen fibers in the Stroma. These data increase the knowledge about the human cornea layers’ ultrastructures and adds new information about its biophysical properties.

Alleviation of the Genotoxic Effect of Experimental Circulatory Brain Hypoxia in Rat Cornea under the Effect of Infrared Low-Level Laser Radiation.

Experimental modeling of oxidative stress was conducted by creating circulatory brain hypoxia, accompanied by development of free radical processes leading to genome instability through an example of anterior epithelial layer of rat cornea. We studied the influence of infrared low-level laser radiation (ILLLR) in the therapeutic dose and mode (pulse power 8 W, λ=0.89 μm). Exposure of animals with circulatory brain hypoxia (ischemia) to ILLLR reduced the level of chromosome aberrations in corneal epitheliocytes and led to change in the mitotic index. Correction of the damaging effects of ischemia in the experiment in the absence of mutagenic effect of ILLLR in therapeutic dose and mode of exposure indicates the possibility of applying this physical method for correcting after-effects of oxidative stress during progression of degenerative eye diseases in ophthalmic practice.

Clearance of Gram-Negative Bacterial Pathogens from the Ocular Surface by Predatory Bacteria

It was previously demonstrated that predatory bacteria are able to efficiently eliminate Gram-negative pathogens including antibiotic-resistant and biofilm-associated bacteria. In this proof-of-concept study we evaluated whether two species of predatory bacteria, Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus, were able to alter the survival of Gram-negative pathogens on the ocular surface. Clinical keratitis isolates of Pseudomonas aeruginosa (strain PAC) and Serratia marcescens (strain K904) were applied to the ocular surface of NZW rabbits followed by application of predatory bacteria. At time intervals, surviving pathogenic bacteria were enumerated. In addition, B. bacteriovorus and S. marcescens were applied to porcine organ culture corneas under contact lenses, and the ocular surface was examined by scanning electron microscopy. The ocular surface epithelial layer of porcine corneas exposed to S. marcescens, but not B. bacteriovorus was damaged. Using this model, neither pathogen could survive on the rabbit ocular surface for longer than 24 h. M. aeruginosavorus correlated with a more rapid clearance of P. aeruginosa but not S. marcescens from rabbit eyes. This study supports previous evidence that predatory bacteria are well tolerated by the cornea, but suggest that predatory bacteria do not considerably change the ability of the ocular surface to clear the tested Gram-negative bacterial pathogens from the ocular surface.

Histological Studies on Ageing Changes in the Cornea of Indian Buffaloes (Bubalus bubalis)

Background: Affections of eye commonly encountered in all the species of animals. If these not treated in time, the vision may be hampered, this may impair the physical ability, utility and productivity of animals leading to economic loss to the animal owners. Corneal dystrophies like basement membrane dystrophy, stromal dystrophy, endothelial dystrophy, corneal erosions and corneal ulcerations etc., seen in the aged buffaloes. The current study was carried out to establish basic data and to provide authentic information to the clinicians on ageing changes in eyes of buffaloes.Methods: The study on age related changes in the cornea was conducted on 63 eye balls of locally available buffaloes of different breeds in and around Proddatur. These buffaloes were categorized into 3 groups based on their age i.e., group I (1-5 yrs), group II (6-10 yrs) and group III (11 yrs and above) buffaloes. The eyeballs were isolated and fixed with Davidson’s fluid. The paraffin sections of cornea were subjected for routine histological study.Result: The mean total thickness (µm) of cornea was increased from group I to III animals and it was 747.22±13.57, 853.33±11.05 and 897.22±8.06. Hence, The mean thickness (µm) of all five layers of cornea increased with advancement of age. Superficial corneal epithelial layer was made up of non keratinized stratified squamous epithelium with 6 to 7 layers at early age and 12-14 layers of cells in adult animals. The mean thickness (µm) of corneal epithelium in group I to III animals was 117.04±3.37, 122.59±2.23 and 133.71±3.55 respectively. Bowman’s membrane of cornea was measured 12.97±0.86, 17.22±0.55 and 18.71±0.68 in group I, II and III animals respectively. The mean thickness (µm) of corneal stroma in group I to III animals was 600.75±10.33, 672.22±10.46 and 690.00±5.32 respectively. This was mainly due to progressive increase in number of collagenous lamellae. The keratocytes became thin in old animals. Thickness of posterior band zone (PBZ) of Descemet’s membrane was increased with advancement of age. The mean thickness (µm) of Descemet’s membrane and endothelium together was in group I, II and III animals was 16.48±0.91, 41.29±2.25 and 54.82±1.44 respectively. The corneal endothelial cells were enlarged and increased in size in old buffaloes. The age related pigmentation was also noticed in epithelium and stroma of cornea in animals of group III. Density of sub basal nerve plexuses between the stroma and the corneal epithelium was not altered with advancement of age.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

PurposeSingle cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. MethodsFour human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). ResultsscRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. ConclusionsOur scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

Review on Conventional and Novel Topical Ocular Drug Delivery System

Ocular drug delivery is a very challenging area for ophthalmologists and drug delivery scientists due to the structural and barrier complexity of the eye. Barriers such as different layers of cornea, sclera, conjunctival blood flow, and tear dilution limit the efficacy of drug delivery to the anterior part of the eye in addition to more barriers present to the posterior part. Due to these, scientists have designed and studied various delivery systems to increase drug delivery and treatment efficacy to the eye. Among conventional ocular drug delivery systems, ophthalmic solution or eye drop is widely used and preferred by consumers. Conventional dosage forms available in the market are emulsion, suspension, ointment and polymeric gels. Several ocular formulations such as nano-formulations, liposomes, ocular inserts, and ocular mini-tablets are also being widely studied as future treatments to improve ocular drug delivery and as an alternative to conventional drug delivery. This review intends to summarise several conventional and novel topical formulations for ocular drug delivery.

Advances in the diagnosis and treatment of keratoconus.

Keratoconus had traditionally been considered a rare disease at a time when the imaging technology was inept in detecting subtle manifestations, resulting in more severe disease at presentation. The increased demand for refractive surgery in recent years also made it essential to more effectively detect keratoconus before attempting any ablative procedure. Consequently, the armamentarium of tools that can be used to diagnose and treat keratoconus has significantly expanded. The advances in imaging technology have allowed clinicians and researchers alike to visualize the cornea layer by layer looking for any early changes that might be indicative of keratoconus. In addition to the conventional geometrical evaluation, efforts are also underway to enable spatially resolved corneal biomechanical evaluation. Artificial intelligence has been exploited in a multitude of ways to enhance diagnostic efficiency and to guide treatment. As for treatment, corneal cross-linking treatment remains the mainstay preventive approach, yet the current main focus of research is on increasing oxygen availability and developing new strategies to improve riboflavin permeability during the procedure. Some new combined protocols are being proposed to simultaneously halt keratoconus progression and correct refractive error. Bowman layer transplantation and additive keratoplasty are newly emerging alternatives to conventional keratoplasty techniques that are used in keratoconus surgery. Advances in tissue engineering and regenerative therapy might bring new perspectives for treatment at the cellular level and hence obviate the need for invasive surgeries. In this review, we describe the advances in the diagnosis and treatment of keratoconus primarily focusing on newly emerging approaches and strategies.

Histomorphological study of corneal dystrophies

Introduction: The term Corneal Dystrophy refers to a rare heterogeneous group of genetically determined, bilateral, symmetric, primary diseases, restricted to cornea and not associated with previous ocular inflammation. The dystrophies are classified based on the layer of cornea involved, into superficial, stromal and posterior dystrophies, each of which is genetically determined. Though the clinical features are often characteristic, definitive diagnosis is possible only after histological examination. The management protocols and visual prognosis vary with the underlying conditions. The incidence of the subtypes varies with the geographic locations. Objective: To study the prevalence of various subtypes of Corneal Dystrophies in corneal button specimens obtained after penetrating keratoplasty in a referral ophthalmic institute and to correlate with the patient’s age and sex. Materials and Methods: Corneal button specimens received in the ocular pathology laboratory over a period of five years were reviewed. Histopathological features of Corneal Dystrophies were studied and subcategorised. Cliniopathological analysis was made. Results: Out of 660 corneal biopsies reviewed, 42 cases were of corneal dystrophy. The patients were between 2 and 71 years of age, with 14 males and 28 females. Macular dystrophy was the most common with 20 cases, followed by Congenital Hereditary Endothelial Dystrophy (CHED) and Fuch’s endothelial dystrophy (FECD), with 7 cases each. Epithelial basement membrane dystrophy, Reis Buckler Dystrophy (RBCD), Granular dystrophy were the other types. Conclusion: Histopathological sub categorisation of various types of Corneal Dystrophies not only helps in understanding the prevalence, but also in predicting the genetic link and prognosis of the disease category. Keywords: Cornea, CHED, Dystrophy, Fuch, Macular.

PREPARATION OF ULTRA-THIN DESCEMET’S MEMBRANE GRAFT USING DECELLULARIZED PORCINE CORNEA

A corneal endothelial layer which is the bottom layer of cornea perform a role to maintain a clarity of cornea. When the number of corneal endothelial cell decrease due to aging or hereditary condition, it is hard to restore because its mitosis capacity is limited. As fundamental treatment, a corneal transplantation is conducted widely. However, there are lots of transplantation waiting lists through a shortage of donor cornea. In this study, we optimize a protocol to prepare ultra-thin descemet’s membrane (DM) graft using decellularized porcine cornea. Using fresh porcine eyeball, a cornea is excised including sclera and conjunctiva. With biopsy punch, endothelium, DM including stroma are partially incised. Next, the cornea was decellularized in 0.1% SDS for 5 hours. Then, a graft is detached DM including stroma. Again, the graft is decelluarized in 0.1% SDS for 10hour at room temperature. The graft was dried in dry oven at 40 celsius overnight. The induced pluripotent stem cell derived corneal endothelial cells was reseeded. Finally, a thickness of dried acellular DM graft was 57.6±7.92um and recelluarized wet graft was 99.2±5.98um. The clarity of recellularized DM graft was 91.8±2.38%. Although the future in-vivo trials would be warranted, tissue engineering DM graft using porcine decellularized corneas might be efficient tools to resolve refractory corneal disease. This work was supported by the Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017K1A4A3014959) and NRF-2018R1D1A1B07050014. This work was also supported by the research grant of the Chungbuk National University in 2017.

Adipose-derived mesenchymal stromal cells promote corneal wound healing by accelerating the clearance of neutrophils in cornea

The dome-shaped cornea is a transparent, non-vascularized, and epithelialized highly organized tissue. Physical and chemical injuries may trigger corneal wound healing (CWH) response and result in neovascularization that impairs the visual function. CWH involves not only migration, proliferation, and differentiation of the cells in different layers of cornea, but also the mobilization of immune cells. We demonstrated here that human adipose-derived mesenchymal stromal cells (ADSCs) could effectively inhibit neovascularization during ethanol-induced injury in mouse cornea. Importantly, we found that while neutrophils are essential for CWH, excessive and prolonged neutrophil retention during the granulation stage contributes to neovascularization. ADSCs were found to promote the clearance of neutrophils in the cornea during the granulation stage, likely via increasing the reverse transendothelial cell migration of CXCR4high neutrophils from cornea to the lung. Our results demonstrate that ADSCs are effective in treating CWH-induced neovascularization and modulation of neutrophil clearance could be novel strategies for better vision recovery after injury.

Topical siRNA delivery to the cornea and anterior eye by hybrid silicon-lipid nanoparticles

The major unmet need and crucial challenge hampering the exciting potential of RNAi therapeutics in ophthalmology is to find an effective, safe and non-invasive means of delivering siRNA to the cornea. Although all tissues of the eye are accessible by injection, topical application is preferable for the frequent treatment regimen that would be necessary for siRNA-induced gene silencing. However, the ocular surface is one of the more complex biological barriers for drug delivery due to the combined effect of short contact time, tear dilution and poor corneal cell penetration. Using nanotechnology to overcome the challenges, we developed a unique silicon-based delivery platform for ocular delivery of siRNA. This biocompatible hybrid of porous silicon nanoparticles and lipids has demonstrated an ability to bind nucleic acid and deliver functional siRNA to corneal cells both in vitro and in vivo. Potent transfection of human corneal epithelial cells with siRNA-ProSilic® formulation was followed by a successful downregulation of reporter protein expression. Moreover, siRNA complexed with this silicon-based hybrid and applied in vivo topically to mice eyes penetrated across all cornea layers and resulted in a significant reduction of the targeted protein expression in corneal epithelium. In terms of siRNA loading capacity, system versatility, and potency of action, ProSilic provides unique attributes as a biodegradable delivery platform for therapeutic oligonucleotides.