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.

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.

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.

UNASSIGNED: This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

OBJECTIVE: 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)).
CONCLUSIONS: 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.

OBJECTIVE: 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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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.