Corneal Epithelium Research Articles

IC3D Classification of Corneal Dystrophies-Edition 3.

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. 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)]. 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. The IC3D third edition provides a current summary of corneal dystrophy information. The article is available online at https://corneasociety.org/publications/ic3d .

Attach importance to the orderly development of different corneal transplant procedures

There are various surgical methods for corneal transplantation, each requiring precise treatment tailored to the characteristics and different layers of corneal opacity or lesions. These layers include the corneal epithelium, stroma (lamellar), and corneal endothelium, with options for full-thickness (penetrating) corneal transplantation or artificial corneal transplantation. However, a current issue in clinical practice is that, regardless of the degree of corneal disease, the availability of corresponding eye bank support, or surgical conditions, classic penetrating (full-thickness) corneal transplantation is universally performed. Alternatively, there is a trend toward adopting technically demanding procedures such as endothelial transplantation and artificial corneal transplantation. This trend has led to increased postoperative complications and the wastage of corneal donor materials. Choosing the appropriate corneal transplant procedure can offer advantages such as preserving more healthy corneal tissue, conserving corneal donors, facilitating rapid vision recovery, and minimizing the risk of immune rejection. Corneologists need to master the indications for various corneal transplant surgeries and systematically perform different corneal transplant procedures based on the surgeon's skills, hospital conditions, and eye bank conditions. This approach aims to enhance the success rate of the surgery.

Dynamic changes of endothelial and stromal cilia are required for the maintenance of corneal homeostasis.

Primary cilia are distributed extensively within the corneal epithelium and endothelium. However, the presence of cilia in the corneal stroma and the dynamic changes and roles of endothelial and stromal cilia in corneal homeostasis remain largely unknown. Here, we present compelling evidence for the presence of primary cilia in the corneal stroma, both in vivo and in vitro. We also demonstrate dynamic changes of both endothelial and stromal cilia during corneal development. In addition, our data show that cryoinjury triggers dramatic cilium formation in the corneal endothelium and stroma. Furthermore, depletion of cilia in mutant mice lacking intraflagellar transport protein 88 compromises the corneal endothelial capacity to establish the effective tissue barrier, leading to an upregulation of α-smooth muscle actin within the corneal stroma in response to cryoinjury. These observations underscore the essential involvement of corneal endothelial and stromal cilia in maintaining corneal homeostasis and provide an innovative strategy for the treatment of corneal injuries and diseases.

Effect of human lactoferrin solution on histological changes of rabbit cornea in alkaline burns

BACKGROUND: Eye burns are a severe injury, accounting for 6.1–38.4% of all ophthalmic diseases. Alkaline burns are the most common and most severe forms of eye burns.
 AIM: Evaluation of the effectiveness of drug topical application based on human lactoferrin in alkaline cornea burns of the 3rd degree in the experiment.
 MATERIAL AND METHODS: The study was conducted on 42 rabbits. It included two groups: the first (without treatment) — alkaline burns during therapy with water for injection (1 drop 3 times a day) — 21 rabbits, the second (experimental) — treatment with lactoferrin solution (concentration 2.5 mg/ml, 1 drop 3 times a day) — 21 rabbits. Alkaline burns were induced by applying a filter paper disc moistened with 5% sodium hydroxide solution to the cornea. 6 eyes of 3 intact animals served as controls. The effectiveness of the drug was evaluated by the rate of closure of the corneal epithelium defect, the time of suppression of the inflammatory reaction in the area of the defect and the limbus, the degree of restoration of the morphological structure of the cornea, as close as possible to the normal rabbit cornea. The obtained data were processed using the methods of variation statistics, the Statistica 10.0 software package. The significance of differences was assessed by calculating the median and interquartile interval. The critical level of significance for statistical criteria was taken as p=0.05.
 RESULTS: From the 3rd day of the study, in the experimental treatment group, there was an acceleration of reepithelialization, restoration of the cornea's own substance, and a more rapid subsidence of inflammation compared to the control group. The thickness of the cornea in the center of the defect in the group without treatment was significantly higher than the values of intact animals at all periods of observation: on the 3rd day after the burn it was 615.99 [450.70–794.07] µm (p=0.000574), reached the maximum by day 7 — it was 1363.16 [907.78–1543.44] µm (p=0.000091), and by day 28 it decreased to 384.38 [376.03–398.14] µm (p=0.0000041). In the group with experimental treatment, the thickness of the cornea in the center of the defect also increased relative to intact animals, starting from the 1st day of pathology, reaching maximum values on the 3rd day — 436.70 [415.57–489.90] µm (p=0.005589). The use of lactoferrin solution in comparison with the first group led to a significant decrease in the thickness of the cornea in the center of the defect on 7th (p=0.039985) and 28th days (p=0.0443).
 CONCLUSION: Local application of lactoferrin solution in alkaline burns of the cornea promotes faster regeneration of the epithelium and restoration of the stroma structure of the rabbit cornea.

Challenges of obtaining cultured corneal endothelial cells for regenerative purposes

Human posterior corneal epithelium (corneal endothelium) has limited proliferative activity both in vivo and in vitro. Disease or dysfunction in these cells leads to impaired corneal transparency of varying degrees of severity, up to blindness. Currently, the only effective standard treatment for corneal endothelial dysfunction is transplantation of donor cornea that contains a pool of healthy and functionally active cells. However, there is a global shortage of donor corneas, which has led to an unmet clinical need and the fact that only 1 patient out of 10 in need receives surgical treatment. Therefore, creation of cellular constructs and artificial human corneas containing healthy endothelium is a very urgent challenge facing modern ophthalmic transplantology. This review presents the current state of affairs, challenges and prospects for obtaining cultured corneal endothelial cells (CECs) in vitro for transplantation purposes.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

The evolution of artificial tears based on hyaluronic acid

In recent years, there has been growing interest in the use of tear substitutes, based on natural polysaccharides in the treatment of dry eye syndrome, the leader of which is hyaluronic acid (HA). It has sufficient biocompatibility, non-immunogenicity, high viscoelasticity, hydrophilic, mucus-adhesive and good moisturizing properties. At the same time, in order to improve artificial tear preparations, there is a need to improve the mechanical and rheological properties of HA, its hygroscopicity, swelling in an aqueous environment and reducing the rate of biodegradation. One of the ways to solve this problem was the chemical modification of HA, by cross-linking its chains with two or more covalent bonds, with the participation of various polyfunctional molecules: urea, HA-cysteine ethyl ester, polyfunctional diepoxides, glutaraldehyde, carbodiimide, and many others. At the same time, the rigidity of the three-dimensional polymer network increases, increasing its resistance to enzymatic decomposition at the site of burial. Cross-linked HA has a higher viscosity compared to native hyaluronic acid due to the binding of its chains, which determines longer retention on the corneal epithelium and naturally makes it possible to reduce the frequency of drug instillations. Convincing data were obtained on the effectiveness of the resulting cross-linked hydrogel with non-Newtonian properties in vitro – on cultures of corneal epithelial cells and in vivo – on models of mechanical trauma and chemical burns of the cornea, as well as in animals with torpid corneal ulcers. The clinical effectiveness of cross-linked hyaluronic acid in the treatment of patients with dry eye syndrome of varying severity, including those due to Sjögren’s syndrome, has also been established. The higher effectiveness of cross-linked HA compared to native HA preparations has been convincingly proven. Taking into account the available information, an official drug has been developed based on cross-linked 0.2% hyaluronic acid Ocutears® Hydro+ (Santen).

Silicone oil as a corneal lubricant to reduce corneal edema and improve visualization during vitrectomy.

To evaluate the efficacy and safety of silicone oil (SO) as a corneal lubricant to improve visualization during vitrectomy. Patients who underwent vitreoretinal surgery were divided into two groups. Group 1 was operated on with initial SO (Oxane 5700) as a corneal lubricant. Group 2 was operated on with initial lactated ringer's solution (LRS) and then replaced with SO as required. Fundus clarity was scored during the surgery. Fluorescein staining was performed to determine the damage to corneal epithelium. Totally 114 eyes of 114 patients were included. Single SO use maintained a clear cornea and provided excellent visualization of surgical image. In group 1, the fundus clarity was grade 3 in 41/45 eyes and grade 2 in 4/45 eyes. In group 2, corneal edema frequently occurred after initial LRS use. The fundus clarity was grade 3 in 19/69 eyes, 2 in 37/69 eyes and 1 in 13/69 eyes (P<0.05). SO was applied in 29 eyes of initial LRS use with subsequent corneal edema, which eliminated the corneal edema in 26 eyes. Corneal fluorescein staining score in group 1 was 0 in 28 eyes, 1 in 11 eyes and 2 in 6 eyes, and 40, 20 and 9, respectively, in group 2 (all P>0.05). The use of SO as a corneal lubricant is effective and safe for preserving and improving corneal clarity and providing clear surgical field during vitrectomy.

Corneal acetylcholine regulates sensory nerve activity via nicotinic receptors

PurposeSensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea. MethodsLocalization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [3H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs. ResultsChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [3H] acetylcholine was synthesized from [3H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist. ConclusionsThe corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

Expression of the SARS-CoV-2 Receptor ACE2 and Protease TMPRSS2 in Ocular Hypertension Eyes of Nonhuman Primate and Human

Purpose Coronavirus disease 2019 is a disease caused by the novel severe acute respiratory syndrome coronavirus 2. The double-positive of angiotensin-converting enzyme 2 and transmembrane protease serine type 2 have a higher risk of being infected by severe acute respiratory syndrome coronavirus 2. The susceptibility of coronavirus disease 2019 in patients with chronic diseases, especially in different tissues of ocular hypertension eyes like glaucoma, is not yet known. Methods An ocular hypertension model was established by laser photocoagulation in rhesus monkeys. The expression of angiotensin-converting enzyme 2 and transmembrane protease serine type 2 in three ocular hypertension eyes and the three control eyes were analyzed using immunofluorescence. Results No difference was observed between ocular hypertension and control eyes in the expression of angiotensin-converting enzyme 2 and transmembrane protease serine type 2 in the conjunctival epithelium, corneal epithelium, and ciliary muscle. In ocular hypertension eyes and control eyes, angiotensin-converting enzyme 2 and transmembrane protease serine type 2 expression were both observed in the retina. Angiotensin-converting enzyme 2 staining of retinal ganglion cells was found to be significantly higher in ocular hypertension eyes than in control eyes. However, there was no difference in angiotensin-converting enzyme 2 and transmembrane protease serine type 2 expression in retinal vessels and choroidal vessels between ocular hypertension and control eyes. In our study, the expression and distribution of angiotensin-converting enzyme 2 and TMPREE2 in human retina were similar to that of non-human primates as expected. Conclusion Our study confirmed that angiotensin-converting enzyme 2 and transmembrane protease serine type 2 were expressed widely in rhesus monkey eyes. When compared with controls eyes, the expression of angiotensin-converting enzyme 2 was higher in the retinal ganglion cells in ocular hypertension eyes, suggesting that high ocular pressure may affect the patients’ ocular susceptibility to severe acute respiratory syndrome coronavirus 2 infection.

The effect of incomplete blinking rate on corneal epithelial remodeling after trans-PRK surgery: a six-month study.

To evaluate the impact of incomplete blink rate on corneal epithelial thickness after transepithelial photorefractive keratectomy (Trans-PRK) surgery. Trans-PRK patients were divided into two groups based on preoperative incomplete blinking rates, namely rates ≤0.5 (41 right eyes, group A) and rates >0.5 (65 right eyes, group B). We used anterior segment optical coherence tomography to measure the corneal epithelial thickness (CET) and lipiview to measure the number of blinks, incomplete blinking rate, and lipid layer thickness (LLT). In both groups, CET decreased at 1 week and 1 month, and the thicknesses in the IT, T, and ST regions exceeded the preoperative levels. Three months after Trans-PRK, the thickness exceeded the preoperative levels and continued to increase during subsequent follow-ups. The blinking times and LLT in both groups decreased at 1 week and gradually increased but did not return to preoperative levels. Group A maintained an incomplete blinking rate of over 0.5 at all postoperative periods. The incomplete blinking rate of group B remained above 0.5 postoperatively, although there was an improvement compared with preoperative rates. Both groups showed a correlation between changes in CET and incomplete blinking rate. There was a correlation between changes in LLT and the incomplete blinking rate after surgery. Both groups showed uneven corneal epithelia thickening, which became more pronounced approaching the peripheral areas. In addition, changes in CET after surgery were positively correlated with the incomplete blinking rate in both patient groups. There was a negative correlation between postoperative LLT and incomplete blinking rate.

Mouse Model of Nitrogen Mustard Ocular Surface Injury Characterization and Sphingolipid Signaling.

Vesicating chemicals like sulfur mustard (SM) or nitrogen mustard (NM) can cause devastating damage to the eyes, skin, and lungs. Eyes, being the most sensitive, have complicated pathologies that can manifest immediately after exposure (acute) and last for years (chronic). No FDA-approved drug is available to be used as medical counter measures (MCMs) against such injuries. Understanding the pathological mechanisms in acute and chronic response of the eye is essential for developing effective MCMs. Here, we report the clinical and histopathological characterization of a mouse model of NM-induced ocular surface injury (entire surface) developed by treating the eye with 2% (w/v) NM solution for 5 min. Unlike the existing models of specific injury, our model showed severe ocular inflammation, including the eyelids, structural deformity of the corneal epithelium and stroma, and diminished visual and retinal functions. We also observed alterations of the inflammatory markers and their expression at different phases of the injury, along with an activation of acidic sphingomyelinase (aSMase), causing an increase in bioactive sphingolipid ceramide and a reduction in sphingomyelin levels. This novel ocular surface mouse model recapitulated the injuries reported in human, rabbit, and murine SM or NM injury models. NM exposure of the entire ocular surface in mice, which is similar to accidental or deliberate exposure in humans, showed severe ocular inflammation and caused irreversible alterations to the corneal structure and significant vision loss. It also showed an intricate interplay between inflammatory markers over the injury period and alteration in sphingolipid homeostasis in the early acute phase.

Effect of Topical Programmed Death-Ligand1 on Corneal Epithelium in Dry Eye Mouse.

Dry eye disease (DED) is a growing health concern that impacts millions of individuals every year, and is associated with corneal injury, excessive oxidative stress and inflammation. Current therapeutic strategies, including artificial tears and anti-inflammatory agents, are unable to achieve a permanent clinical cure due to their temporary nature or adverse side effects. Therefore, here, we investigated the effectiveness of the topical administration of programmed death-ligand 1 (PD-L1) in the mouse model of DED. The model was generated in C57BL/6 mice by excising the extra orbital lacrimal gland and causing desiccation stress with scopolamine injections. Subsequently, either phosphate-buffered saline (3 µL/eye) or PD-L1 (0.5 µg/mL) was topically administered for 10 days. Tear volume was evaluated with phenol red thread, and corneal fluorescein staining was observed to quantify the corneal epithelial defect. Corneas were collected for histological analysis, and the expression levels of inflammatory signaling proteins such as CD4, CD3e, IL-17, IL-1β, pIkB-α, pNF-kB and pERK1/2 were assessed through immunofluorescence and Western blot techniques. Our results demonstrate that desiccating stress-induced corneal epithelial defect and tear secretion were significantly improved by topical PD-L1 and could reduce corneal CD4+ T cell infiltration, inflammation and apoptosis in a DED mouse model by downregulating IL-17 production and ERK1/2-NFkB pathways.

Multilayered-Amniotic Membrane Transplantation in Gunshot Powder Injury: A Case Report

Background: Amniotic membrane transplantation has been widely used to repair various ocular surface conditions, including inflammatory, infectious, traumatic, and neoplastic conditions, with satisfactory results. a case of a 34-year-old male hit by a gunpowder explosion.&#x0D; Case presentation: We reported a case of a 34-year-old male hit by a gunpowder explosion. The right eye was hyperemic and felt blurry after the incident. On examination of the right eye, visual acuity was 20/150, and foreign bodies and erosion were found in the cornea. The patient planned to do foreign body extraction followed by a multilayered-amniotic membrane transplantation (ML-AMT) procedure. On the first and second day of follow-up, on the right eye, the visual acuity was 1/300, palpebral edema was decreased, and subconjunctival hemorrhage was found. On the cornea, there were ML-AMT and bandage contact lenses.&#x0D; Conclusion: Gunshot powder injury in the eyes is quite rare. The damage that can occur ranges from corneal epithelium abrasion to perforation and rupture of the eyeball. AMT has been successfully used in the management of eye trauma with defects on the ocular surface.

Matrix-Assisted Cell Transplantation for the Treatment of Limbal Stem Cell Deficiency in a Rabbit Model.

With the development of regenerative medicine in ophthalmology, the identification of cells with high proliferative potential in the limbal area has attracted the attention of ophthalmologists and offered a new option for treatment in clinical practice. Limbal stem cell deficiency (LSCD) is an identified eye disease with a difficult and negative outcome, for which the traditional treatment is keratoplasty. This study sought to evaluate the efficacy of matrix-assisted cell transplantation consisting of in vitro-cultured autologous limbal stem cells (LSCs) and type I collagen for the treatment of LSCD in rabbits. LSCD was induced in 10 rabbits by a combination of mechanical limbectomy and alkali burns. Cells were cultured on a plate for 14 days before being transferred to a collagen-based matrix for another 7 days. Rabbits were divided into two groups as follows: the experimental group (five rabbits) received matrix-assisted cell transplantation, while the control group (five rabbits) received only conservative therapy with anti-inflammatory eye drops. During the postoperative period, all rabbits were examined using slit-lamp biomicroscopy with photo-registration and fluorescent staining, impression cytology and anterior segment optical coherence tomography (AS-OCT). Rabbits were euthanized at 30 and 120 days, and their corneas were processed for histology and immunohistochemistry. As a consequence, rabbits in the experimental group demonstrated the restoration of the corneal epithelium and transparency without epithelial defects. Moreover, goblet cells were absent in the central zone of the corneal epithelium. In conclusion, our new method of treatment enhanced the corneal surface and is an effective method of treatment for LSCD in rabbits.

Corneal Epithelial Thickness Mapping: A Major Review.

The corneal epithelium (CE) is the outermost layer of the cornea with constant turnover, relative stability, remarkable plasticity, and compensatory properties to mask alterations in the underlying stroma. The advent of quantitative imaging modalities capable of producing epithelial thickness mapping (ETM) has made it possible to characterize better the different patterns of epithelial remodeling. In this comprehensive synthesis, we reviewed all available data on ETM with different methods, including very high-frequency ultrasound (VHF-US) and spectral-domain optical coherence tomography (SD-OCT) in normal individuals, corneal or systemic diseases, and corneal surgical scenarios. We excluded OCT studies that manually measured the corneal epithelial thickness (CET) (e.g., by digital calipers) or the CE (e.g., by confocal scanning or handheld pachymeters). A comparison of different CET measuring technologies and devices capable of producing thickness maps is provided. Normative data on CET and the possible effects of gender, aging, diurnal changes, refraction, and intraocular pressure are discussed. We also reviewed ETM data in several corneal disorders, including keratoconus, corneal dystrophies, recurrent epithelial erosion, herpes keratitis, keratoplasty, bullous keratopathy, carcinoma in situ, pterygium, and limbal stem cell deficiency. The available data on the potential role of ETM in indicating refractive surgeries, planning the procedure, and assessing postoperative changes are reviewed. Alterations in ETM in systemic and ocular conditions such as eyelid abnormalities and dry eye disease and the effects of contact lenses, topical medications, and cataract surgery on the ETM profile are discussed.

Transmembrane Protein CMTM6 Alleviates Ocular Inflammatory Response and Improves Corneal Epithelial Barrier Function in Experimental Dry Eye.

To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms. CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis. CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells. The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.

3D printing sequentially strengthening high-strength natural polymer hydrogel bilayer scaffold for cornea regeneration.

3D printing of high-strength natural polymer biodegradable hydrogel scaffolds simultaneously resembling the biomechanics of corneal tissue and facilitating tissue regeneration remains a huge challenge due to the inherent brittleness of natural polymer hydrogels and the demanding requirements of printing. Herein, concentrated aqueous solutions of gelatin and carbohydrazide-modified alginate (Gel/Alg-CDH) are blended to form a natural polymer hydrogel ink, where the hydrazides in Alg-CDH are found to form strong hydrogen bonds with the gelatin. The hydrogen-bonding-strengthened Gel/Alg-CDH hydrogel demonstrates an appropriate thickened viscosity and shear thinning for extrusion printing. The strong hydrogen bonds contribute to remarkably increased mechanical properties of Gel/Alg-CDH hydrogel with a maximum elongation of over 400%. In addition, sequentially Ca2+-physical crosslinking and then moderately chemical crosslinking significantly enhance the mechanical properties of Gel/Alg-CDH hydrogels that ultimately exhibit an intriguing J-shaped stress-strain curve (tensile strength of 1.068 MPa and the toughness of 677.6 kJ/m2). The dually crosslinked Gel-Alg-CDH-Ca2+-EDC hydrogels demonstrate a high transparency, physiological swelling stability and rapid enzymatic degradability, as well as suturability. The growth factor and drug-loaded biomimetic bilayer hydrogel scaffold are customized via a multi-nozzle printing system. This bioactive bilayer hydrogel scaffold considerably promotes regeneration of corneal epithelium and stroma and inhibits cornea scarring in rabbit cornea keratoplasty.

Assessing progression limits in different grades of keratoconus from a novel perspective: precision of measurements of the corneal epithelium

BackgroundTo assess repeatability and reproducibility of corneal epithelium thickness (ET) measured by a spectral-domain optical coherence tomographer (SD-OCT)/Placido topographer (MS-39, CSO, Florence, Italy) in keratoconus (KC) population at different stages, as well as to determine the progression limits for evaluating KC progression.MethodsA total of 149 eyes were enrolled in this study, with 29 eyes in the forme fruste keratoconus (FFKC) group, 34 eyes in the mild KC group, 40 eyes in the moderate KC group, and 46 eyes in the severe KC group. Employing the within-subject standard deviation (Sw), test-retest variability (TRT), coefficient of variation (CoV), and intraclass correlation coefficient (ICC) to evaluate intraoperator repeatability and interoperator reproducibility.ResultsThe repeatability and reproducibility of MS-39 in patients with KC were acceptable, according to ICC values ranging from 0.732 to 0.954. However, patients with more severe KC and progressive peripheralization of the measurement points had higher TRTs but a thinning trend. The current study tended to set the cut-off values of mild KC, moderate KC, and severe KC to 4.9 µm, 5.2 µm, and 7.4 µm for thinnest epithelium thickness (TET). When differences between follow-ups are higher than those values, progression of the disease is possible. As for center epithelium thickness (CET), cut-off values for mild KC, moderate KC, and severe KC should be 2.8 µm, 4.4 µm, and 5.3 µm. This might be useful in the follow-up and diagnosis of keratoconus.ConclusionsThis study demonstrated that the precision of MS-39 was reduced in measuring more severe KC patients and more peripheral corneal points. In determining disease progression, values should be differentiated between disease-related real changes and measurement inaccuracies. Due to the large difference in ET measured by MS-39 between various stages of disease progression, it is necessary to accurately grade KC patients to avoid errors in KC clinical decision-making.

Corneal and epithelial thickness mapping in diabetic patients using optical coherence tomography

Purpose To evaluate corneal and epithelial thickness using the anterior segment optical coherence tomography and to correlate these changes with major disease parameters, that is duration of the disease, status of control (glycated hemoglobin level), and stage of diabetic retinopathy. Patients and methods Corneal and epithelial thickness profile of 25 patients diagnosed with diabetes mellitus and 25 healthy individuals were measured by anterior segment optical coherence tomography. Full ophthalmological examination of all patients was done. The disease duration and the glycated hemoglobin levels were recorded. Results In diabetic patients, there was nonsignificant thinning in mean central epithelial thickness (P=0.14). The mean central corneal thickness is slightly thicker, however, it did not reach significance (P=0.051). There was statistically significant thickening in central stromal thickness (P=0.015). There was a significant negative correlation between the severity of retinopathy and superior epithelial thickness (P=0.037). The duration of the disease showed significant correlation with most of epithelial thickness values, however, HA1c level was not found to have any statistically significant effect on corneal and epithelial parameters. Conclusion Chronic metabolic stress of diabetes has an impact on corneal epithelium and stroma. Severity of diabetic retinopathy should be considered as a risk factor during corneal surgeries to avoid epithelial and stromal-associated complications.