Central Corneal Epithelium Research Articles

PAX6-WNK2 Axis Governs Corneal Epithelial Homeostasis.

Limbal stem/progenitor cells (LSCs) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. A previous study revealed that paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CECs). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation. An air-liquid culture system was used to differentiate LSCs into mature CECs. Specific targeting PAX6 short-hairpin RNAs were used to knock down PAX6 in LSC. RNA sequencing (RNA-seq) was used to analyze shPAX6-transfected CECs and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases. WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers. As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.

Urolithin A alleviates cell senescence by inhibiting ferroptosis and enhances corneal epithelial wound healing.

To analyze the therapeutic effect and mechanism of Urolithin A (UA) on delayed corneal epithelial wound healing. The C57BL/6 mice were continuously exposed to hyperosmotic stress (HS) for 7 days followed by the removal of central corneal epithelium to establish a delayed corneal epithelial wound healing model in vivo. In vitro, the human corneal epithelial cell line (HCE-T) was also incubated under HS. UA was administered in vivo and in vitro to study its effects on corneal epithelial cells. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to detect the level of cell senescence. Transcriptome sequencing (RNA-seq) was conducted to elucidate the molecular mechanism underlying the effect of UA on corneal epithelial repair. Additionally, the expression of senescence-related and ferroptosis-related genes and the levels of lipid peroxides (LPO) and malondialdehyde (MDA) were measured. Hyperosmotic stress (HS) significantly increased the proportion of SA-β-gal staining positive cells in corneal epithelial cells and upregulated the expression of p16 and p21 (p < 0.0001). Topical application of UA decreased the accumulation of senescent cells in corneal epithelial wounds and promoted epithelial wound healing. The results of RNA-seq of HS-induced corneal epithelial cells showed that the ferroptosis pathway was significantly dysregulated. Further investigation revealed that UA decreased the level of oxidative stress in HCE-T cells, including the levels of LPO and MDA (p < 0.05). Inhibition of ferroptosis significantly prevented cellular senescence in HS-induced HCE-T cells. In this study, UA promoted HS-induced delayed epithelial wound healing by reducing the senescence of corneal epithelial cells through the inhibition of ferroptosis.

Intraoperative variability of corneal epithelium thickness in photorefractive keratectomy

PurposeTo evaluate the intraoperative central corneal epithelial thickness (ET) as measured by optical coherence pachymetry (OCP) in myopic eyes undergoing alcohol-assisted photorefractive keratectomy (PRK).MethodsA 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.ResultsThe 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)).ConclusionThe 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.

Isolation and characterization of rabbit limbal niche cells

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs.Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRβ) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRβ and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRβ in rabbit-LNCs P4.In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRβ+).

Long-Term Effects of Adipose-Derived Stem Cells for the Treatment of Bilateral Limbal Stem Cell Deficiency

Purpose To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). Methods A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. Results No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). Conclusion ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

Efficacy of Topical Timolol Eye Drops in Treatment of Myopic Regression After Small Incision Lenticule Extraction: A Case Report

Purpose: To describe a patient with high myopia who experienced slight myopic regression twice during follow-up after small incision lenticule extraction (SMILE), which can be eased by topical treatment of timolol. Methods: Case report. Results: Six months after a SMILE procedure for high myopia in both eyes, the patient's uncorrected distance visual acuity (UDVA) was 0.8 and both eyes lost one line compared to preoperative corrected distance visual acuity (CDVA) by myopic shift, which was accompanied by central corneal epithelial thickening. After 2 months of treatment with topical timolol eye drops, the UDVA improved two more lines with flattened corneal curvature and thinned epithelial thickness. After 5 months without timolol treatment, he lost two lines of UDVA again with thicker epithelial thickness. After another 2 months of treatment with timolol, the epithelial thickness could be seen to have thinned month by month, and the UDVA improved two or more lines. There was no notable myopic shift in both eyes for the next 3 months with medication withdrawal. During the whole follow-up period, the change in corneal and epithelial thickness remarkably fluctuated with the timolol treatment, but the posterior corneal curvature was stable. Conclusions: This case report describes refractive regression that can repeatedly occur in the middle and long term after a SMILE procedure for high myopia, which could be correlated with the thickening of the central corneal epithelium. Treatment with timolol eye drops can help to improve visual acuity by thinning the compensated epithelial thickness to flatten corneal curvature. [ Journal of Refractive Surgery Case Reports. 2023;3(4):e47–e52.]

Effect of PKH-26-labeled exosomes derived from bone marrow mesenchymal stem cells on corneal epithelium regeneration in diabetic mice.

It is known that bone marrow mesenchymal stem cells (BM-MSCs) could speed up the regeneration of diabetic corneal epithelium. To investigate the effect of exosomes derived from mouse BM-MSCs on corneal epithelium regeneration in diabetic mice. Diabetic mouse models were established using streptozotocin (STZ), and their central corneal epithelium was scratched under a microscope. The diabetic mice were randomly divided into three groups: the control group was injected with subconjunctival phosphate buffer saline; the exosomes group was treated with a subconjunctival injection of exosomes derived from BM-MSCs; and the BM-MSCs group was treated with a subconjunctival injection of BM-MSCs. The corneal epithelium repair rates in the three groups were compared, and the distribution of the exosomes derived from BM-MSCs labeled with PKH-26 was observed by immunofluorescence. Hematoxylin-eosin staining of the corneal tissue was observed 72 h after the treatments in the three groups. The diabetic mice were successfully established by a blood glucose level >16.7 mmol/L after 8 weeks. The corneal epithelium healing rates in experimental groups 1 and 2 were significantly higher than those of the control group at 24, 48, and 72 h (P<0.05). However, there was no significant difference in the corneal epithelial healing rate between experimental groups 1 and 2 (P>0.05). The exosomes derived from BM-MSCs were found in the superficial corneal stroma in experimental groups 1 and 2, with the majority of the exosomes distributed in the limbal epithelium at the edge of the injury area. The proliferation of corneal epithelial cells in experimental groups 1 and 2 was more obvious than that in the control group. The exosomes derived from BM-MSCs labeled with PKH-26 significantly promoted the repair of corneal epithelial injury in diabetic mice. These exosomes might be a substitute for BM-MSCs in the repair of diabetic keratopathy, suggesting a new idea for the repair of diabetic keratopathy with "cell-free" stem cell therapy, which will require a clinical study.

Role of corneal epithelial thickness during myopic regression in femtosecond laser-assisted in situ keratomileusis and transepithelial photorefractive keratectomy

The study aimed to investigate the relationship between changes in corneal epithelial thickness and the outcome of myopic regression after femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and transepithelial photorefractive keratectomy (TPRK). This study included 45 eyes of 25 patients undergoing FS-LASIK and 44 eyes of 24 patients undergoing TPRK. Myopic regression occurred in these patients postoperatively from 8 to 21 months. The corneal epithelial thickness was measured using a spectral-domain optical coherence tomography at the onset of regression, 3 months after treatment, and 3 months after drug withdrawal. Compared with that of preoperation, corneal epithelial thickness increased when regression occurred in both groups (all P < 0.05). The thickness of central corneal epithelium in FS-LASIK and TPRK groups reached 65.02 ± 4.12µm and 61.63 ± 2.91µm, respectively. The corneal epithelial thickness decreased when myopic regression subsided after 3 months of steroid treatment compared to the onset (P < 0.05). With a decrease in corneal epithelial thickness, the curvature of the anterior corneal surface, central corneal thickness, and refractive power all decreased (all P < 0.05). The corneal epithelial thickness and refractive error remained relatively stable after 3 months of treatment withdrawal (P > 0.05). The corneal epithelial thickness determined the outcome of myopic regression similarly in FS-LASIK and TPRK. When the corneal epithelium thickened, regression occurred. After steroid treatment, epithelial thickness decreased whereas regression subsided.

Healing Ability of Central Corneal Epithelium in Rabbit Ocular Surface Injury Models.

PurposeWound healing of the corneal epithelium mainly involves two types of cells: limbal stem/progenitor cells (LSCs) and differentiated central corneal epithelial cells (CECs). The healing ability of CECs is still debatable, and its correlated transcriptomic alterations during wound healing are yet to be elucidated. This study aimed to determine the healing ability and mechanisms underlying the actions of CECs using rabbit ocular surface injury models.MethodsA central corneal ring-like residual epithelium model was used to investigate the healing ability of CECs. Uninjured and injury-stimulated LSCs and CECs were collected for transcriptomic analysis. The analysis results were verified by quantitative reverse transcriptase polymerase chain reaction, immunofluorescence staining, and two types of rabbit corneal injury models.ResultsDuring wound healing, the upregulated genes in LSCs were mostly enriched in the mitotic cell cycle–related processes, but those in CECs were mostly enriched in cell adhesion and migration. CECs could repair the epithelial defects successfully at one-time injuries. However, after repetitive injuries, the CECs repaired notably slower and failed to completely heal the defect, but the LSCs repaired even faster than the one-time injury.ConclusionsOur results indicated rabbit CECs repair the epithelial defect mainly depending on migration and its proliferative ability is limited, and LSCs are the main source of regenerative epithelial cells.Translational RelevanceThis study provides information on gene expression in the corneal epithelium during wound healing, indicating that regulation of the cell cycle, cell adhesion, and migration may be the basis for future treatment strategies for corneal wound healing.

Defining an Optimal Sample Size for Corneal Epithelial Immune Cell Analysis Using in vivo Confocal Microscopy Images.

PurposeIn vivo confocal microscopy (IVCM) images are frequently used to quantify corneal epithelial immune cell (IC) density in clinical studies. There is currently limited evidence to inform the selection of a representative image sample size to yield a reliable IC density estimate, and arbitrary numbers of images are often used. The primary aim of this study was to determine the number of randomly selected, unique IVCM images required to achieve an acceptable level of accuracy when quantifying epithelial IC density, in both the central and peripheral cornea. The secondary aim was to evaluate the consistency and precision of an image selection approach where corneal epithelial IC density was quantified from “three representative images” selected independently by three experienced observers.MethodsAll combinations of two to 15 non-overlapping IVCM images were used for deriving IC density estimates, for both the central and peripheral cornea, in 20 healthy participants; the density value from averaging quantifications in the 16 images was defined as the “true mean”. IC density estimates were compared with the true mean in each corneal region using a mean ratio. Intraclass correlation coefficients (ICCs) were used to evaluate the consistency of the mean ratios of IC density estimates derived from the method involving the manual selection of “three representative images” by the observers. The precision of the IC density estimates was compared to a scenario involving three randomly selected images.ResultsA total of 12 randomly selected, non-overlapping IVCM images were found to be required to produce a corneal epithelial IC density estimate that was within 30% of the true mean, 95% of the time, for the central cornea; seven such images produced an equivalent level of precision in the peripheral cornea. Mean ratios of corneal IC density estimates derived from “three representative images” methods had poor consistency between observers (ICC estimates <0.5) and similar levels of precision when compared with using three randomly selected images (p > 0.05 for all comparisons), in both the central and peripheral cornea.ConclusionsData presented in this study can inform image selection methods, and the sample size required for a preferred level of accuracy, when quantifying IC densities in the central and peripheral corneal epithelium using IVCM images.

Post-Ortho-K Corneal Epithelium Changes in Myopic Eyes.

The study is aimed at evaluating corneal epithelial thickness changes associated with overnight orthokeratology (ortho-K). In this retrospective study, epithelial thickness was measured using optical coherence tomography (OCT) before and after 1 day, 1 week, 1 month, and 3 months ortho-K nightly lens wear. Compared with pre-orthokeratology measurements, central (2 mm) corneal epithelium thickness was significantly reduced at 1 day, 1 week, 1 month, and 3 months with ortho-K (P < 0.05). Paracentral (2 mm~5 mm annular ring) epithelial thickness was also significantly reduced at superior temporal, inferior temporal, temporal, and inferior locations after ortho-K (P < 0.05), while midperipheral (5 mm~6 mm annular ring) epithelial thickness was greater post- than pre-ortho-K at superior, superior temporal, inferior temporal, inferior, and inferior nasal locations (P < 0.05). In other zones, superior, superior nasal, nasal, and inferior nasal in paracentral annular ring and temporal and superior nasal in midperipheral ring, epithelial thickness underwent no significant change. Ortho-K lens wear caused the central corneal epitheliums to thin. The temporal half zones become thinner in paracentral zones and thicker in midperipheral zones.

Uzun Süreli Bilgisayar Kullanıcılarında Kuru Göz Parametreleri, Gözyaşı Menisküs Yüksekliği ve Kornea Epitel Kalınlığının Kısa Süreli Hafta İçi Çalışma Periyodundaki Değişimi: Kohort Çalışması

Objective: To investigate the changes in dry eye parameters, tear meniscus height and epithelial thickness over a shortterm period (working week) in long-term computer users. Material and Methods: There were 46 eyes of 23 patients with dry eye symptoms who used computers for at least 6 h per day were included in this study. The Schirmer's test scores, tear break-up time (TBUT), ocular surface disease index (OSDI) score, epithelial thickness and tear meniscus levels by spectral domain-optical coherence tomography were recorded. All measurements of the patients were repeated on Monday morning before starting work (first visit) and on Friday of the same week after work (second visit) without medication. Results: The mean TBUT values were 12.41±2.68 s, 9.69±2.39 s, the Schirmer's test values were 20±5.47 mm, 11.89±3.62 mm (p<0.001); the tear meniscus levels were 158.65±41.18 μm, 118.15±31.99 μm (p<0.001); the OSDI scores were 12.13±4.89, 18±5.50 (p<0.001); and the central corneal epithelium thicknesses were 33.5±5.81 μm, 33.19±6.10 μm (p=0.703) upon the first and second visit, respectively. There was no significant difference in central corneal epithelial thickness (CET) between the two visits (p=0.703). Conclusion: While changes in the OSDI score, the TBUT, the Schirmer's test score and the tear meniscus levels were observed in long-term computer users in the short-term working week period, there was no short-term effect on central CET.

Epithelial cells removed in advanced surface ablation (ASA) surgery can be used as a source of corneal samples to perform in vitro studies

Human corneal epithelial cells are needed to study corneal pathophysiology in vitro. Due to the limitations of cell lines, the use of primary cells is highly desirable, but the scarcity of human tissues, along with ethical issues, make it difficult to accomplish all required experiments. In advanced surface ablation (ASA), the central corneal epithelium is removed and discarded. We hypothesized that ASA samples could be used to perform in vitro assays. In this study, 29 samples from patients undergoing ASA were recovered in supplemented DMEM/F12 culture medium, RIPA buffer, or RLT lysis buffer. The first aim was to determine whether cells could be maintained in culture. Although with the explant technique, tissue pieces did not attach to the culture surface, after disaggregation, cells showed high viability (90.0 ± 6.0%), attached to plates, and remained viable for up to 14 days. The second aim was to elucidate if ASA samples could be used to study protein or gene expression. Cytokeratin-3, ZO-1, Ki67, and E-cadherin protein expression were confirmed by immunofluorescence. Total protein (485.8 ± 115.8 μg) was isolated from cells in RIPA buffer, and GAPDH was detected by Western blotting, indicating that samples are adequate for protein studies. RNA (9.0 ± 3.6 μg) was isolated from samples in RLT lysis buffer, and GAPDH gene expression was studied by PCR, confirming that samples were also suitable for gene expression studies. These results suggest that samples obtained from corneal surface ablation procedures may constitute a valuable source of human cells to accomplish in vitro studies.

A new approach for quantifying epithelial and stromal thickness changes after orthokeratology contact lens wear.

The aim of the study was to develop an automatic segmentation approach to optical coherence tomography (OCT) images and to investigate the changes in epithelial and stromal thickness profile and radius of curvature after the use of orthokeratology (Ortho-K) contact lenses. A total of 45 right eyes from 52 participants were monitored before, and after one month of, uninterrupted overnight Ortho-K lens wear. The tomography of their right eyes was obtained using optical OCT and rotating Scheimpflug imaging (OCULUS Pentacam). A custom-built MATLAB code for automatic segmentation of corneal OCT images was created and used to assess changes in epithelial thickness, stromal thickness, corneal and stromal profiles and radii of curvature before, and after one month of, uninterrupted overnight wear of Ortho-K lenses. In the central area (0–2 mm diameter), the epithelium thinned by 12.8 ± 6.0 µm (23.8% on average, p < 0.01) after one month of Ortho-K lens wear. In the paracentral area (2–5 mm diameter), the epithelium thinned nasally and temporally (by 2.4 ± 5.9 µm, 4.5% on average, p = 0.031). The stroma thickness increased in the central area (by 4.8 ± 16.1 µm, p = 0.005). The radius of curvature of the central corneal anterior surface increased by 0.24 ± 0.26 mm (3.1%, p < 0.01) along the horizontal meridian and by 0.34 ± 0.18 mm (4.2%, p < 0.01) along the vertical meridian. There were no significant changes in the anterior and posterior stromal radius of curvature. This study introduced a new method to automatically detect the anterior corneal surface, the epithelial posterior surface and the posterior corneal surface in OCT scans. Overnight wear of Ortho-K lenses caused thinning of the central corneal epithelium. The anterior corneal surface became flattered while the anterior and posterior surfaces of the stroma did not undergo significant changes. The results are consistent with the changes reported in previous studies. The reduction in myopic refractive error caused by Ortho-K lens wear was mainly due to changes in corneal epithelium thickness profile.

Impact of Different Oxygen Supply Methods on the Healing of Corneal Epithelial Wound and the Level of Acetylcholine.

Purpose To investigate the impact of different oxygen supply methods on corneal epithelial wound healing and acetylcholine level during wound healing. Methods We randomly divided 75 rabbits into three groups: A, B, and C, with 25 rabbits in each group. The central corneal epithelium was removed from all eyes of the rabbits using a 5 mm trephine. Group A rabbits were given low flow oxygen (3 L/min; concentration: 33%) for 2 h per day through goggles. Group B rabbits were given low flow oxygen (3 L/min; concentration: 33%) for 2 h per day via oxygen masks for inhalation. Group C rabbits healed naturally. The area of healed corneal epithelium and acetylcholine content in corneal epithelium were determined at 12 h, 24 h, and 36 h after injury. Results At 12 h, 24 h, and 36 h after injury, the healing area of corneal epithelium in the three groups was in the order group A > group B > group C (P < 0.05). At all timepoints, the acetylcholine level in corneal epithelium was in the order of group A > group B > group C (P < 0.05). In all three groups, the acetylcholine content in corneal epithelium showed the order 12 h > 24 h > 36 h (P < 0.05). There was a correlation between acetylcholine expression and the area of unhealed corneal epithelium, and the correlation coefficients of groups A, B, and C were 0.80, 0.83, and 0.85 respectively. Conclusion Increasing oxygen concentration through inhalation or via goggles can promote corneal epithelial wound healing, but increasing local oxygen concentration of the eye showed a better effect. Acetylcholine may play an important role in the early process of corneal epithelial wound healing.

A pilot study investigating the effect of extended contact lens wear on limbal and central corneal morphology

PurposeTo investigate the effect of long-term extended soft contact lens wear on limbal and central corneal cell morphology, and limbal architecture. MethodsEach participant attended a study visit involving in vivo confocal microscopy of central corneal and limbal epithelium. Scans were graded by five masked graders for three features: central epithelial irregularity, limbal epithelial irregularity and the prominence of palisades of Vogt. The variability of grades between different graders and the difference of grades between extended wearers and daily soft/non-contact lens wearers were assessed. ResultsNineteen participants (9 extended soft contact lens wearers and 10 daily soft/non-contact lens wearers) aged 31–65 years were enrolled in this study. Scans from 37 eyes were included in the analysis. Agreement between graders for each feature was moderate to good with inter class correlation >0.7. While there were no significant differences in central epithelial cell irregularity (p = 0.527) and the prominence of palisade of Vogt (p = 0.182) between extended or daily soft/non-contact lens wearers, limbal epithelial cell irregularity showed a trend with increased irregularity in extended soft contact lens wearers (p = 0.091). ConclusionsWhile no differences in limbal cell morphology and structureor central epithelial cell wasfound in thissubjective grading study of extended wearers compared to daily soft/non-contact lens wearers, further studies using a larger sample size or a longitudinal study design are warranted.

Topical Delivery of Levocarnitine to the Cornea and Anterior Eye by Thermosensitive in-situ Gel for Dry Eye Disease.

PurposeTo prepare the levocarnitine thermosensitive in situ gel (LCTG) and evaluate its effect on dry eye disease (DED).MethodsDraize eye irritation test and other examinations were used to evaluate the eye irritation after multiple administration of LCTG. The Schirmer test, fluorescein sodium staining, HE staining and TUNEL staining were used to detect the tear secretion, corneal injury, histopathological changes of the cornea and lacrimal gland, and the apoptosis rate of cornea epithelial cells after 3 days of the administration. The conjunctival goblet cell density was detected by PAS staining, and the expression levels of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-9 (MMP-9) of corneal epithelial cells were detected by immunofluorescence staining after 7 days of the administration.ResultsLCTG is non-irritating to rabbit eyes and has good biocompatibility. LCTG administration for 3 days can significantly increase the amount of tear secretion in mice with DED, promote corneal epithelial integrity and central corneal epithelium thickness recovery, and improve the pathological morphology and structure of corneal and lacrimal gland tissues, and reduce the apoptosis rate of the corneal epithelial cells. After 7 days of the administration, the preparation can promote the proliferation of conjunctival goblet cells and down-regulate the cornea expression levels of MMP-3 and MMP-9 in epithelial cells.ConclusionThe LCTG has a good curative effect on mice with DED, and the overall curative effect is better than that of levocarnitine solution.

Corneal epithelium and limbal region alterations due to glaucoma medications evaluated by anterior segment optic coherence tomography: a case-control study

Aim To investigate the corneal epithelial and limbal epithelial alterations in patients under topical glaucoma treatment using anterior segment-OCT (AS-OCT) and to determine the changes of the limbal region due to the preservatives and glaucoma drugs, that can progress to limbal stem cell deficiency (LSCD). Limbal thickness was measured by AS-OCT to evaluate limbal cell deficiency. Methods Forty-seven patients using topical medication for glaucoma, and 48 control subjects were enrolled in this matched case-control study. The patients were divided into four groups according to the treatment regimens. Group 1: One-drug regimen, Group 2: Two-drug regimen, Group 3: Three-drug regimen, Group 4: Four-drug regimen For the ocular surface evaluation; tear break-up time with standard fluorescein sodium sterile strip application, Schirmer test-I, Ocular Surface Disease Index Questionnaire, and AS-OCT were performed. Results A total of 95 subjects were included: 47 eyes of 47 patients with glaucoma medication and 48 eyes of 48 healthy subjects. There was a statistically significant difference between patients and controls according to BUT, SCH, and OSDI (p < 0.001). The mean central corneal epithelium thickness was 48.5 ± 5.3 in patients and 54.5 ± 5.9 in controls (p < 0.001). The mean central total corneal thickness was 529.2 ± 41.2 in patients and 536 ± 35.3 in controls (p = 0.335). The mean limbal epithelium thickness was 64.1 ± 9.1 in patients and 76 ± 11.5 in controls (p < 0.001). Conclusion Using at least one glaucoma drug caused limbal area injury, changed ocular surface measurements, and significantly reduced the limbal epithelial thickness where the stem cells reside. The limbal epithelial thickness measurement by AS-OCT seems to be an innovative, non-invasive, and promising technique for detecting and staging corneal damage in topical glaucoma therapy.

Loss of FOXC1 contributes to the corneal epithelial fate switch and pathogenesis

Forkhead box C1 (FOXC1) is required for neural crest and ocular development, and mutations in FOXC1 lead to inherited Axenfeld–Rieger syndrome. Here, we find that FOXC1 and paired box 6 (PAX6) are co-expressed in the human limbus and central corneal epithelium. Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers. FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2. FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells, but also disrupts the collagen metabolic process and interferon signaling pathways. Loss of interferon regulatory factor 1 and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer. Collectively, our results reveal a FOXC1-mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.

Establishing a Porcine Eye Model for Manual Sub-Bowman Layer Photorefractive Keratomileusis.

Objective To establish a porcine eye model for manual sub-Bowman layer photorefractive keratomileusis (SBPRK), which is a reformed surface ablation refractive surgery that results in preserving the corneal Bowman layer (BL). Methods The SBPRK group consisted of eleven eyes of 8 healthy pigs with BL flaps by mechanical technique followed by laser ablation. Regarding the remaining 5 eyes, 3 random eyes had transepithelium photorefractive keratectomy (TransPRK) (the TransPRK group), while the other 2 eyes were untreated (the blank control group). All the pigs were followed up for 8 weeks. Slit-lamp biomicroscopy and optical coherence tomography (OCT) were examined before the surgeries and at 1 week, 4 weeks, and 8 weeks after the surgeries. Results In a few days after the surgery, 3 eyes of the SBPRK group were excluded from the study because of poor healing of the corneal flaps. At the 1st postoperative week, one eye had an irregular defect of about 3 mm in the central corneal epithelium area; the cornea of the other 7 eyes had just light edema with intact epithelium just like the cornea of the TransPRK group. At the 4th week, in the SBPRK group, the cornea was slightly hazy (haze stage 1). While in the TransPRK group, the cornea was hazier (haze stage 2). At the 8th week, in the SBPRK group, both corneas were almost transparent, and the edges of the BL flaps could not be clearly seen. Meanwhile, in the TransPRK group, the corneal haze became lighter and thinner. OCT showed that, in the SBPRK group, there was high reflection in the BL layer, and it was obvious at 1 week postoperation, decreased at 4 weeks, and calmed down at 8 weeks. However, in the TransPRK group, the high reflection diffused in the anterior corneal stroma at 1 week postoperation, enhanced at 4 weeks, and weakened at 8 weeks. Conclusions Preserving the BL while conducting surface refractive surgery may result in less haze than TransPRK. However, further study is still needed, and this technique still requires refining until it becomes a standard clinical procedure.