Comparison of the repeatability and reproducibility of corneal thickness mapping using optical coherence tomography according to tear film break-up time.

BACKGROUND: To compare the repeatability and reproducibility of corneal and corneal epithelial thickness mapping using anterior segment optical coherence tomography (AS-OCT) according to tear film break-up time (TBUT). METHODS: The included eyes were divided into three subgroups according to TBUT (group 1: TBUT ≤ 5 s, group 2: 5 s < TBUT ≤ 10 s, and group 3: TBUT > 10 s). All eyes were imaged separately thrice by two operators to obtain the thickness maps (TMs) of the cornea and corneal epithelium based on spatial zones encompassing a 9-mm-diameter area. Each TM consisted of 25 areas. Intraoperator (repeatability) and interoperator (reproducibility) standard deviations (Sws), coefficients of variation (CoVs), and intraclass correlation coefficients (ICCs) among the tests were calculated and compared in all the areas. RESULTS: Altogether, 132 eyes of 67 subjects were included (50, 47, and 35 eyes in groups 1, 2, and 3; respectively). The ICCs of corneal epithelial thickness and corneal thickness were > 0.75 in most of the areas. Pairwise comparisons showed that AS-OCT exhibited lower repeatability in group 1 than in groups 2 and 3 (P < 0.05). However groups 2 and 3 showed similar results. Sws and CoVs of corneal epithelial thickness exhibited no significant interoperator differences. While no significant differences were observed in corneal thickness in most of the areas. CONCLUSIONS: TBUT significantly influences the repeatability of corneal and corneal epithelial thickness measurements. Poor tear film stability requires careful evaluation of corneal epithelial thickness.

Blepharoptosis and corneal epithelial thickness alterations, is there any relation?

BACKGROUND: To compare the epithelial thickness map of ptotic eyes of blepharoptosis patients with contralateral non- ptotic eyes. METHODS: Unilateral blepharoptosis patients were enrolled consecutively. Patients were underwent full ophthalmologic examination and their demographic data such as age and gender and specific ptosis findings e.g. the cause and duration, MRD-1, and levator palpebralis superioris function were registered. Anterior segment imaging for epithelial thickness measurements was done using the Avanti RTVue-XR platform. The corneal epithelial thickness maps of ptotic and non-ptotic eyes were compared. RESULTS: 44 patients with unilateral blepharoptosis were included in the study. 27 (61.4%) of them were female and 17 (38.6%) cases were male. The mean of the patients' ages was 24.40 ± 15.16 years. Ptotic eyes had significantly thinner superior (p = 0.000), superior-temporal (p = 0.000) and superior-nasal (p = 0.005) sectors of the cornea and slightly thicker corneal epithelium (CE) in the inferior-nasal sector. The correlation of difference of superior-inferior CE was evaluated with different parameters including patient's age (p = 0.457), type of blepharoptosis (p = 0.786), duration of blepharoptosis (p = 0.477) and MRD1 (p = 0.248), but no correlation was found. CONCLUSIONS: This study revealed that lid position in blepharoptosis may have effects on the corneal epithelial thickness map. Because of the lower position of upper eyelid, a thinning effect on superior corneal sectors may happen.

Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing.

BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive. METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing. RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing. CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

Characterization of Ocular Morphology in Col4a3-/- Mice as a Murine Model for Alport Syndrome.

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Reticulated Retinoic Acid Synthesis is Implicated in the Pathogenesis of Dry Eye in Aqp5 Deficiency Mice.

Purpose: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Methods: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Results: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Conclusions: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

Ectoine Enhances Mucin Production Via Restoring IL-13/IFN-γ Balance in a Murine Dry Eye Model.

Purpose: This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease. Methods: A dry eye model was established in C57BL/6 mice exposed to desiccating stress (DS) with untreated (UT) mice as controls. DS mice were topically treated with 2.0% ectoine or PBS vehicle. Corneal epithelial defects were assessed by Oregon Green Dextran (OGD) fluorescent staining. Conjunctival goblet cells, ocular mucins, and T help (Th) cytokines were evaluated by immunofluorescent staining or ELISA, and RT-qPCR. Results: Compared with UT mice, corneal epithelial defects were detected as strong punctate OGD fluorescent staining in DS mice with vehicle, whereas ectoine treatment largely reduced OGD staining to near-normal levels. Conjunctival goblet cell density and cell size decreased markedly in DS mice, but was significantly recovered by ectoine treatment. The protein production and mRNA expression of two gel-forming secreted MUC5AC and MUC2, and 4 transmembrane mucins, MUC1, MUC4, MUC16, and MUC15, largely decreased in DS mice, but was restored by ectoine. Furthermore, Th2 cytokine IL-13 was inhibited, whereas Th1 cytokine IFN-γ was stimulated at protein and mRNA levels in conjunctiva and draining cervical lymph nodes (CLNs) of DS mice, leading to decreased IL-13/IFN-γ ratio. Interestingly, 2.0% ectoine reversed their alternations and restored IL-13/IFN-γ balance. Conclusions: Our findings demonstrate that topical ectoine significantly reduces corneal damage, and enhances goblet cell density and mucin production through restoring imbalanced IL-13/IFN-γ signaling in murine dry eye model. This suggests therapeutic potential of natural osmoprotectant ectoine for dry eye disease.

Oridonin ameliorates ocular surface inflammatory responses by inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway in dry eye.

Chronic inflammation is one of the central drivers in the development of dry eye disease (DED), in which pyroptosis induced by the NLRP3/caspase-1/gasdermin D (GSDMD) pathway plays a key role. This pathway has become a major target for the treatment of a variety of inflammatory disorders. Oridonin (Ori) is a naturally occurring substance with anti-inflammatory properties obtained from Rabdosia rubescens. Whether Ori can exert an anti-inflammatory effect on DED, and its anti-inflammatory mechanism of action, are still unknown. This experiment is intended to investigate the impact of Ori on the hyperosmolarity-induced NLRP3/caspase-1/GSDMD pyroptosis pathway in immortalized human corneal epithelial (HCE-T) cells, as well as its efficacy and mechanism of action on ocular surface injury in DED mice. Our study showed that Ori could inhibit hyperosmotic-induced pyroptosis through the NLRP3/caspase-1/GSDMD pathway in HCE-T cells, and similarly, Ori inhibited the expression of this pathway in DED mice. Moreover, Ori was protective against hyperosmolarity-induced HCE-T cell damage. In addition, we found that the morphology and number of HCE-T cells were altered under culture conditions of various osmolarities. With increasing osmolarity, the proliferation, migration, and healing ability of HCE-T cells decreased significantly, and the expression of N-GSDMD was elevated. In a mouse model of DED, Ori application inhibited the expression of the NLRP3/caspase-1/GSDMD pyroptosis pathway, improved DED signs and injury, decreased corneal sodium fluorescein staining scores, and increased tear volume. Thus, our study suggests that Ori has potential applications for the treatment of DED, provides potential novel therapeutic approaches to treat DED, and provides a theoretical foundation for treating DED using Ori.

Extensive Contact Lens Wear Modulates Expression of miRNA-320 and miRNA-423-5p in the Human Corneal Epithelium: Possible Biomarkers of Corneal Health and Environmental Impact.

The identification of new biomarkers of ocular diseases is nowadays of outmost importance both for early diagnosis and treatment. Epigenetics is a rapidly growing emerging area of research and its involvement in the pathophysiology of ocular disease and regulatory mechanisms is of undisputable importance for diagnostic purposes. Environmental changes may impact the ocular surface, and the knowledge of induced epigenetic changes might help to elucidate the mechanisms of ocular surface disorders. In this pilot study, we investigated the impact of extensive contact lens (CL) wearing on human corneal epithelium epigenetics. We performed ex vivo analysis of the expression of the miR-320 and miR-423-5p involved in the processes of cellular apoptosis and chronic inflammation. The human corneal epithelium was harvested from healthy patients before the photorefractive keratectomy (PRK). The patients were divided into two age- and sex-matched groups accordingly to CL wearing history with no CL wearers used as a control. The epithelium was stored frozen in dry ice at -80 °C and forwarded for miRNA extraction; afterwards, miRNA levels were detected using real-time PCR. Both miRNAs were highly expressed in CL wearers (p < 0.001), suggesting epigenetic modifications occurring in chronic ocular surface stress. These preliminary results show the relationships between selected miRNA expression and the chronic ocular surface stress associated with extensive CL use. MicroRNAs might be considered as biomarkers for the diagnosis of ocular surface conditions and the impact of environmental factors on ocular surface epigenetic. Furthermore, they might be considered as new therapeutic targets in ocular surface diseases.

The Role of Ubiquitination and the E3 Ligase Nedd4 in Regulating Corneal Epithelial Wound Healing.

Purpose: Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH. Methods: Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4. Results: Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4. Conclusions: Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.

Intraoperative variability of corneal epithelium thickness in photorefractive keratectomy.

PURPOSE: To evaluate the intraoperative central corneal epithelial thickness (ET) as measured by optical coherence pachymetry (OCP) in myopic eyes undergoing alcohol-assisted photorefractive keratectomy (PRK). METHODS: A retrospective review of patients who underwent alcohol-assisted PRK was performed. Data were abstracted on age, gender, contact lens (CL) wear, preoperative refractive errors, keratometry, topographic and ultrasonic pachymetry, and intraoperative OCP measurements before and after epithelium removal. The central ET was calculated by subtracting OCP measurement after epithelium removal from the OCP measurement prior to epithelium removal. RESULTS: The study comprised of 162 consecutive eyes from 81 patients. Mean age was 26.73 ± 6.47 years, 50.6% were males. CL was used in 92 eyes (56.8%). The mean sphere and spherical equivalent were -3.60 ± 1.84 D and -3.26 ± 1.85D, respectively. The mean intraoperative ET was 58.22 ± 17.53 µm (range, 15-121µm). Fifty-five percent of the eyes had an ET measurement above or below the range of 40-60µm. ET was significantly higher in the second operated eye compared to the first operated eye (p = 0.006), and an association was found to CL-wear (p = 0.03). There was no significant difference in thickness between genders (p = 0.62), and no correlation to patient age (p = 0.45, rp = 0.06), refractive errors (p > 0.30,rp=-0.07-0.08), nor keratometry(p > 0.80, rp=-0.01- (-0.02)). CONCLUSION: The intraoperative assessment of ET in alcohol-assisted PRK showed a high variability of the central corneal epithelium, with a significant difference between the first and second operated eyes. This difference may have implications when the epithelium is not included in the surgical planning in surface ablation.

In vitro and in vivo studies of ocular topically administered NLC for the treatment of uveal melanoma.

Uveal melanoma is one of the most common and aggressive intraocular malignancies, and, due to its great capability of metastasize, it constitutes the most incident intraocular tumor in adults. However, to date there is no effective treatment since achieving the inner ocular tissues still constitutes one of the greatest challenges in actual medicine, because of the complex structure and barriers. Uncoated and PEGylated nanostructured lipid carriers were developed to achieve physico-chemical properties (mean particle size, homogeneity, zeta potential, pH and osmolality) compatible for the ophthalmic administration of (S)-(-)-MRJF22, a new custom-synthetized prodrug for the potential treatment of uveal melanoma. The colloidal physical stability was investigated at different temperatures by Turbiscan® Ageing Station. Morphology analysis and mucoadhesive studies highlighted the presence of small particles suitable to be topically administered on the ocular surface. In vitro release studies performed using Franz diffusion cells demonstrated that the systems were able to provide a slow and prolonged prodrug release. In vitro cytotoxicity test on Human Corneal Epithelium and Human Uveal Melanoma cell lines and Hen's egg-chorioallantoic membrane test showed a dose-dependent cytotoxic effect of the free prodrug on corneal cells, whose cytocompatibility improved when encapsulated into nanoparticles, as also confirmed by in vivo studies on New Zealand albino rabbits. Antiangiogenic capability and preventive anti-inflammatory properties were also investigated on embryonated eggs and rabbits, respectively. Furthermore, preliminary in vivo biodistribution images of fluorescent nanoparticles after topical instillation in rabbits' eyes, suggested their ability to reach the posterior segment of the eye, as a promising strategy for the treatment of choroidal uveal melanoma.

Histology, histochemistry and ultrastructure of cornea of domestic pigs (Sus scrofa domesticus).

A comprehensive light and ultrastructural examination of the cornea in Domestic Pigs (Sus scrofa domesticus) revealed four distinct layers: the anterior epithelium, corneal stroma, Descemet's membrane and endothelium. Although Bowman's layer was not distinctly identified through histology, histochemical analysis indicated the presence of a rudimentary Bowman's layer, possibly vestigial from evolution. Scanning electron microscopy of the outer corneal surface unveiled two cell types, characterized by micro-projections, with light cells exhibiting shorter, thicker projections compared to dark cells. Examination of the inner surface via scanning electron microscopy demonstrated an endothelial layer devoid of cilia and microvilli, yet faint round to oval elevations were observed, potentially representing cell nuclei. Transmission electron microscopy unveiled that basal cells of the anterior epithelium closely adhered to the basement membrane, featuring half desmosomes along the basal surface. These basal cells extensively interconnected through interdigitations and a few desmosomes. The superficial cell layer consisted of a few rows of closely attached flat cells, forming a leak-proof layer with zona occludens. The outermost cells of this layer displayed fine projections to enhance the surface area, facilitating tear film distribution. At lower magnification, Transmission electron microscopy of the corneal stroma revealed alternating light and dark bands, with light bands representing transverse sections of collagen fibril lamellae and dark bands corresponding to longitudinal or oblique sections. Spindle-shaped keratocytes (fibroblasts) were identified as the primary stromal cells, intermingled between the lamellae, and featured long processes in close contact with neighbouring keratocytes. Overall, the histomorphology of the pig cornea resembles that of the human cornea except indistinct Bowman's membrane. This detailed understanding of the normal corneal structure in pigs hold great significance for biomedical research, providing a valuable reference for studies involving this animal model.

Epithelial thickness map-adjusted transepithelial photorefractive keratectomy for treatment of myopic astigmatism: 12-month results.

PURPOSE: To evaluate the refractive results of Transepithelial Photorefractive Keratectomy (t-PRK) with the Technolas Teneo2 Excimer laser platform. METHODS: In this retrospective comparative interventional case series, a total of 199 patients with myopia ranging from - 1 to - 7 diopters were enrolled and separated into three groups based on their target refraction of zero (group 1), - 0.25 (group 2), and - 0.5 diopters (group 3), respectively. The main outcome measure was post-operative cycloplegic refraction. Trans-PRK was performed using the Technolas Teneo2 Excimer laser. To prevent any remaining epithelium during stromal ablation, we adopted the thickest epithelial point in the 7 mm central map as the reference for Phototherapeutic keratectomy (PTK) depth. Patients were examined three and twelve months after the procedure, and the results were analyzed. RESULTS: At the 12-month follow-up, uncorrected distance visual acuity was 20/20 in all patients. However, there was a significant difference in cycloplegic spherical equivalent refraction between the three groups. The 12-month post-operative spherical equivalent refraction was 0.90 ± 0.33 D, 0.79 ± 0.26 D, and 0.60 ± 0.19 D in groups 1, 2, and 3, respectively (P < 0.001; Kruskal-Wallis test). The rates of spherical equivalent refraction of more than 0.75 D were 58.3%, 39.1%, and 9.1% in the 0 D, - 0.25 D, and -0.50 D groups, respectively (P < 0.001; Chi-squared test). CONCLUSIONS: The t-PRK with Technolas Teneo2 Excimer laser and epithelial thickness map adjustment of PTK induce a significant amount of residual hyperopia (> 0.75 D) in a large proportion of eyes with a target refraction of 0 or - 0.25, which is significantly reduced by using a target refraction of - 0.5.

Construction of Dome-Shaped 3D Corneal Epithelial Tissue Models Based on Eyeball-Shaped Gel Microspheres.

By overcoming interspecies differences and mimicking the in vivo microenvironment, three-dimensional (3D) in vitro corneal models have become a significant novel tool in contemporary ophthalmic disease research. However, existing 3D corneal models struggle to replicate the actual human corneal environment, especially the dome-shaped physiological structure with adjustable curvature. Addressing these challenges, this study introduces a straightforward method for fabricating collagen/chitosan-alginate eyeball-shaped gel microspheres with a Janus structure via a two-phase aqueous system, used subsequently to construct in vitro 3D corneal epithelial tissue models. By adjusting the diameter ratio of collagen/chitosan to alginate droplets, we can create eyeball-shaped gel microspheres with varying curvatures. Human corneal epithelial cells were seeded on the surfaces of these microspheres, leading to the formation of in vitro 3D corneal epithelial tissues characterized by dome-like multilayers and tight junctions. Additionally, the model demonstrated responsiveness to UVB exposure through the secretion of reactive oxygen species (ROS) and proinflammatory factors. Therefore, we believe that in vitro 3D corneal epithelial tissue models with dome-shaped structures hold significant potential for advancing ophthalmic research.

Mitochondria Transplantation Promotes Corneal Epithelial Wound Healing.

Purpose: The integrity of the corneal epithelium is essential in maintaining normal corneal function. Conditions disrupting the corneal epithelial layer range from chemical burns to dry eye disease and may result in impairment of both corneal transparency and sensation. Identifying factors that regulate corneal wound healing is key for the development of new treatment strategies. Here, we investigated a direct role of mitochondria in corneal wound healing via mitochondria transplantation. Methods: Human corneal epithelial cells (hCECs) were isolated from human corneas and incubated with mitochondria which were isolated from human ARPE-19 cells. We determined the effect of mitochondria transplantation on wound healing and proliferation of hCECs. In vivo, we used a mouse model of corneal chemical injury. Mitochondria were isolated from mouse livers and topically applied to the ocular surface following injury. We evaluated the time of wound repair, corneal re-epithelization, and stromal abnormalities. Results: Mitochondria transplantation induced the proliferation and wound healing of primary hCECs. Further, mitochondria transplantation promoted wound healing in vivo. Specifically, mice receiving mitochondria recovered twice as fast as control mice following corneal injury, presenting both enhanced and improved repair. Corneas treated with mitochondria demonstrated the re-epithelization of the wound area to a multi-layer appearance, compared to thinning and complete loss of the epithelium in control mice. Mitochondria transplantation also prevented the thickening and disorganization of the corneal stromal lamella, restoring normal corneal dehydration. Conclusions: Mitochondria promote corneal re-epithelization and wound healing. Augmentation of mitochondria levels via mitochondria transplantation may serve as an effective treatment for inducing the rapid repair of corneal epithelial defects.

Fosfenopril Attenuates Inflammatory Response in Diabetic Dry Eye Models by Inhibiting the TLR4/NF-κB/NLRP3 Signaling Pathway.

Purpose: The purpose of this study was to investigate the involvement of the TLR4/NF-κB/NLRP3 signaling pathway and its underlying mechanism in diabetic dry eye. Methods: Two models of diabetic dry eye were established in high glucose-induced human corneal epithelial (HCE-T) cells and streptozotocin (STZ)-induced C57BL/6 mice, and the TLR4 inhibitor fosfenopril (FOS) was utilized to suppress the TLR4/NF-κB/NLRP3 signaling pathway. The expression changes in TLR4, NF-κB, NLRP3, and IL-1ß, and other factors were detected by Western blot and RT‒qPCR, the wound healing rate was evaluated by cell scratch assay, and the symptoms of diabetic mice were evaluated by corneal sodium fluorescein staining and tear secretion assay. Results: In the diabetic dry eye model, the transcript levels of TLR4, NF-κB, NLRP3, and IL-1ß were raised, and further application of FOS, a TLR4 inhibitor, downregulated the levels of these pathway factors. In addition, FOS was found to be effective in increasing the wound healing rate of high glucose-induced HCE-T cells, increasing tear production, and decreasing corneal fluorescence staining scores in diabetic mice, as measured by cell scratch assay, corneal sodium fluorescein staining assay, and tear production. Conclusions: The current study found that the TLR4/NF-κB/NLRP3 signaling pathway regulates diabetic dry eye in an in vitro and in vivo model, and that FOS reduces the signs of dry eye in diabetic mice, providing a new treatment option for diabetic dry eye.

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

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

Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

Severe corneal injury can lead to blindness even after prompt treatment. 14-3-3zeta, a member of an adaptor protein family, contributes to tissue repair by enhancing cellular viability and inhibiting fibrosis and inflammation in renal disease or arthritis. However, its role in corneal regeneration is less studied. In this study, filter disc of 2-mm diameter soaked in sodium hydroxide with a concentration of 0.5 N was placed at the center of the cornea for 30 s to establish a mouse model of corneal alkali injury. We found that 14-3-3zeta, which is mainly expressed in the epithelial layer, was upregulated following injury. Overexpression of 14-3-3zeta in ocular tissues via adeno-associated virus-mediated subconjunctival delivery promoted corneal wound healing, showing improved corneal structure and transparency. In vitro studies on human corneal epithelial cells showed that 14-3-3zeta was critical for cell proliferation and migration. mRNA-sequencing in conjunction with KEGG analysis and validation experiments revealed that 14-3-3zeta regulated the mRNA levels of ITGB1, PIK3R1, FGF5, PRKAA1 and the phosphorylation level of Akt, suggesting the involvement of the PI3K-Akt pathway in 14-3-3zeta-mediated tissue repair. 14-3-3zeta is a potential novel therapeutic candidate for treating severe corneal injury.

Transporter Protein Expression of Corneal Epithelium in Rabbit and Porcine: Evaluation of Models for Ocular Drug Transport Study.

The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.

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

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