How to reduce conjunctival cyst formation after strabismus surgery?

PURPOSE: To determine our rate of conjunctival cyst developing after strabismus surgery and to minimize its formation. MATERIALS AND METHODS: The data of 4026 eyes of 2662 patients were included in our retrospective analysis of, those that underwent strabismus surgery at the Beyoglu Eye Training and Research Hospital Strabismus Unit between 2015 and 2021. Clinical characteristics and follow-up data of patients were recorded together with age, sex, and operation type. All patients were examined postoperatively on 1st day, 1st month and 6th month. RESULTS: The mean age of the patients was 15.66 ± 11.81 (1-59 years). While two eyes were operated on in 1494 patients, one eye was operated on in 1168 patients. The surgeries performed were recession and/or resection of the horizontal and vertical rectus muscles and inferior and superior oblique procedures. The conjunctival cyst was detected in 11 patients postoperatively between 20 days and 8 months. The cyst was detected on the nasal side in 7 cases and temporal in 4. CONCLUSION: Conjunctival cyst is a rare complication after strabismus surgery and it is thought to be caused by a proliferation of the conjunctival epithelium. In addition to proper closure of the conjunctiva, attention to personal hygiene, avoidance of contacts that may increase suture contamination, and attention to the fact that the operation period is not long are actions that can help prevent conjunctival cyst formation.

Strengthening of the barrier function in human telomerase reverse transcription (hTERT) immortalized corneal and conjunctival epithelium by double-stranded RNA.

To investigate the response to polyinosinic:polycytidylic acid [poly(I:C)], a double-stranded RNA Toll-like receptor 3 agonist that mimics viral infection, in the barrier function of two established human telomerase reverse transcriptase-immortalized cell lines, termed HCLE for the human corneal-limbal epithelial line and HCjE for the human conjunctival-epithelial line. In this study, HCLE and HCjE cells were used to evaluate the underlying mechanism of epithelial-cell barrier function regulation. Briefly, HCLE and HCjE cells were first cultured on 12-well Transwell® (Corning®) filter-plates, and reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical examinations were then performed to assess tight junction (TJ)-related protein expression and cellular distribution. Next, the barrier function of the cells was measured via transepithelial electrical resistance (TEER) and paracellular molecular flux. The cells were then stimulated with poly(I:C) and the TEER and TJ-related protein expressions were analyzed. Similar to that in in vivo epithelium, the expression of claudin (CLDN) subtypes CLDN-1, -4, and -7 was observed in the HCLE and HCjE cells, and the barrier function in the HCLE cells was tighter than that in the HCjE cells. Post stimulation with poly(I:C), TEER of the HCLE and HCjE cells increased in a dose- and time-dependent manner, the production of TJ-related protein mRNA and CLDN-4 protein were elevated, and the barrier function of the HCLE and HCjE cells increased, thus possibly indicating that the increased barrier function is a defense mechanism against viral infection.

Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

Conjunctival epithelial hyperplasia in a patient with a nodular lesion in the palpebral conjunctiva: A case report.

The conjunctiva is a thin and delicate mucous membrane lining the inner eyelid and the anterior surface of the eyeball. Although hyperplastic changes can occur due to nonspecific chronic inflammation, 'conjunctival epithelial hyperplasia' has not been sufficiently established as a pathological entity. Additionally, the immunohistochemical (IHC) features of both the intact conjunctiva epithelium and conjunctival epithelial hyperplasia have not been sufficiently evaluated. The present report describes the case of an 86-year-old man who consulted with an ophthalmologist for a 6-month-old nodular lesion on his left eye. Located in the medial aspect of the left lower palpebral conjunctiva, the lesion was slightly erythematous and smooth. An excisional biopsy of the lesion was performed to obtain a pathological diagnosis. The hematoxylin and eosin sections revealed a thickened conjunctival epithelium composed of hyperplastic cuboidal epithelial cells and goblet cells, indicating conjunctival epithelial hyperplasia. Atypia, increased mitosis and a papillomatous architecture, indicative of neoplastic changes, were not observed. This resulted in conjunctival squamous intraepithelial neoplasia and squamous cell papilloma being ruled out. IHC analysis was performed to further characterize the lesion as well as the intact conjunctival epithelium. The thick conjunctival epithelium was composed of epithelial cells that stained positive for cytokeratin [AE1/AE3 (intensity: +), CK5/6 (intensity: ++), and CK7 (intensity: +)] and p63-positive basal cells (intensity: +) whose presence in the conjunctiva has received insufficient recognition. Moreover, squamous metaplasia was found in a segment of the thick conjunctiva, which exhibited IHC features similar to those of hyperplasia. CK5/6 was positive, indicating endogenous squamous differentiation of the conjunctival epithelial hyperplasia. These findings led to the diagnosis of conjunctival epithelial hyperplasia as a pathological entity. Further collection and analysis of several cases of conjunctival epithelial hyperplasia may lead the development of preventative methods and drug treatments for this lesion, and additional prognostic data, such as the recurrence rate.

Tryptophan as a Safe Compound in Topical Ophthalmic Medications: In Vitro and In Vivo Studies.

BACKGROUND: To evaluate the effects of tryptophan (TRP) on normal human corneal and conjunctival epithelium in vitro and the re-epithelization of corneal erosion in rabbits. MATERIALS AND METHODS: Corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) cultures were used. The cellular metabolism, viability, secretion of IL-1ß, IL-6, IL-10, cytoskeleton organization, transwell migration were determined. Cells were incubated in the presence of TRP at 1-100 µM. After corneal de-epithelization rabbits received TRP drops (100 µM), 5 times a day. RESULTS: TRP increased conjunctival epithelium metabolism at 50 µM and increased the viability of corneal epithelium at 100 µM. TRP (10 µM) enhanced the production of IL-6 by the corneal epithelium and had no effect on IL-1ß and IL-10. CONCLUSIONS: TRP had no influence on the cellular cytoskeleton but induced a significant pseudopodia projection in both epithelia. TRP did not influence corneal re-epithelization in vivo. TRP was not toxic for corneal and conjunctival epithelia.

Is there a role for tapered topical dose steroidal treatment for dry eye disease? A randomized, pilot study.

PURPOSE: To evaluate the effect of tapered doses of loteprednol-etabonate in dry eye disease patients. MATERIALS AND METHODS: Dry eye and treatment outcomes were assessed by Schirmer I test, tear BUT, lissamine green conjunctival staining, fluorescein corneal staining, and HLA-DR expression on conjunctival cells. Patients received either loteprednol-etabonate 0.5% twice daily for 14 days tapered to once daily for 14 days, and then twice weekly for 28 days (n = 10), or NaCl 0.9%. RESULTS: A significant decrease of ocular surface inflammation and improvement of symptoms was recorded in the study group compared with controls at days 14 and 56. Change from baseline in HLA-DR expression in CD45+ conjunctival cells was significantly higher in treated patients at day 14. Intraocular pressure and best corrected visual acuity were preserved in all treated eyes. CONCLUSIONS: Tapered doses of loteprednol etabonate 0.5% suspension controlled ocular surface inflammation, improving dry eye symptoms.

Kynurenic Acid Accelerates Healing of Corneal Epithelium In Vitro and In Vivo.

Kynurenic acid (KYNA) is an endogenous compound with a multidirectional effect. It possesses antiapoptotic, anti-inflammatory, and antioxidative properties that may be beneficial in the treatment of corneal injuries. Moreover, KYNA has been used successfully to improve the healing outcome of skin wounds. The aim of the present study is to evaluate the effects of KYNA on corneal and conjunctival cells in vitro and the re-epithelization of corneal erosion in rabbits in vivo. Normal human corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) lines were used. Cellular metabolism, cell viability, transwell migration, and the secretion of IL-1ß, IL-6, and IL-10 were determined. In rabbits, after corneal de-epithelization, eye drops containing 0.002% and 1% KYNA were applied five times a day until full recovery. KYNA decreased metabolism but did not affect the proliferation of the corneal epithelium. It decreased both the metabolism and proliferation of conjunctival epithelium. KYNA enhanced the migration of corneal but not conjunctival epithelial cells. KYNA reduced the secretion of IL-1ß and IL-6 from the corneal epithelium, leaving IL-10 secretion unaffected. The release of all studied cytokines from the conjunctival epithelium exposed to KYNA was unchanged. KYNA at higher concentration accelerated the healing of the corneal epithelium. These favorable properties of KYNA suggest that KYNA containing topical pharmaceutical products can be used in the treatment of ocular surface diseases.

Ozone-Based Eye Drops Activity on Ocular Epithelial Cells and Potential Pathogens Infecting the Front of the Eye.

Confirmation of the biological effectiveness of new ophthalmic preparations introduced in the market is an important element in maintaining the safety of using this type of medications. This study aimed to investigate the activity of Ozodrop® on human corneal and conjunctival epithelial cells, as well as its antibacterial and antifungal activity. Cytotoxicity analyses of ocular surface epithelial cells were performed in vitro by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Neutral Red uptake assays. The level of nitric oxide released by the cells was assessed by the Griess method. The reduction of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical by the tested formulation was analyzed. Microbiological tests were also performed. It was found that the Ozodrop® preparation exhibited biological activity, but was less active than the reference antibiotics and the anti-yeast agent. The cytotoxic activity of the Ozodrop® formulation was dependent on the time of cell exposure to it. No toxic effect was observed in the short-term, for up to 3 h. It appeared after 24 h of exposure of the cells to the preparation. The drops showed antioxidant activity in the specified concentration range. They also stimulated the release of nitric oxide, mainly by corneal epithelial cells. The Ozodrop® formulation exhibits biological activity that can be considered useful in the treatment of infections in the front part of the eye.

Generation of functional conjunctival epithelium, including goblet cells, from human iPSCs.

The conjunctival epithelium, which covers the sclera (the white of the eye) and lines the inside of the eyelids, is essential for mucin secretion and the establishment of a healthy tear film. Here, we describe human conjunctival development in a self-formed ectodermal autonomous multi-zone (SEAM) of cells that were derived from human-induced pluripotent stem cells (hiPSCs) and mimic whole-eye development. Our data indicate that epidermal growth factor (EGF) drives the generation of cells with a conjunctival epithelial lineage. We also show that individual conjunctival cells can be sorted and reconstituted by cultivation into a functional conjunctival epithelium that includes mucin-producing goblet cells. Keratinocyte growth factor (KGF), moreover, is necessary for the maturation of hiPSC-derived conjunctival epithelium-particularly the goblet cells-indicating key complementary roles of EGF and KGF in directing the differentiation and maturation, respectively, of the human conjunctival epithelium.

sPLA2-IIa participates in ocular surface inflammation in humans with dry eye disease.

PURPOSE: To determine the roles of secretory phospholipase A2-IIa (sPLA2-IIa) in the inflammatory responses of the compromised ocular surface. METHODS: Conjunctival impression cytology (IC) samples and tears were collected from patients with mild to severe non-Sjogren's dry eye disease (DED) and normal controls. The IC samples were analyzed for transcription of sPLA2-IIa and inflammatory cytokine/chemokine genes using quantitative real-time RT-PCR (qRT2-PCR) and pathway-focus PCR-array. The tear samples were analyzed for 13 inflammatory cytokines and chemokines with Millipore 13-Plex kit. Finally, sPLA2-IIa-treated human conjunctival epithelial cell (HCjE) cultures were analyzed with a pathway-focused PCR array. RESULTS: Transcription of sPLA2-IIa was significantly increased in severe DED patients as compared to those of mild DED patients and normal controls. The transcription of inflammatory cytokines (IL-1ß, IL-4, IL-6, IL-17, TNF-α, IFN-γ), chemokines (IL-8, CXCL10, CXCL11, CXCL-14, CCR6, LTB) and matrix metalloproteinase 9 (MMP9) were simultaneously increased in the same IC samples of DED. Concentrations of IL-6 and IL-8 in tears were significantly higher in DED patients than those of the controls and positively correlated to DED severity scores. On the other hand, IL-2, IL-4, IL-10, IL-12 and IFN-γ were significantly lower in DED patients than those in the controls and inversely correlated to DEWS scores. Single treatment of sPLA2-IIa, IL-1ß or TNF-α of HCjE cells induced minimal to no PGE2 production. When sPLA2-IIa was added to HCjE cells that were pre-treated with pro-inflammatory cytokines (TNF-α or IL-1ß), significant stimulation of PGE2 production was observed, concurrent with the extensive transcriptional changes of many inflammatory cytokines/chemokines and their receptors. CONCLUSION: sPLA2-IIa activity was elevated and not only associated with inflammatory changes in DED patient samples, but was also found to cooperate with TNF- α and IL-1ß to induce inflammatory response in human conjunctival epithelial cells. Understanding the roles of sPLA2-IIa in ocular surface inflammation may lead to better strategies for the treatment of chronic inflammation associated with DED and other ocular inflammatory conditions.

Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva.

PURPOSE: The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC). METHODS: PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells. RESULTS: From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva. CONCLUSION: Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

Assessment of goblet cell size and density in relation to epithelial cell (multi)layering on conjunctival impression cytology samples.

PURPOSE: To assess goblet cell size and numbers in relation to the extent of multilayering of conjunctival impression cytology (CIC) samples as a basis for reducing variability in image selection for goblet cell density (GCD) estimates. METHODS: CIC was undertaken immediately postmortem off the superior bulbar conjunctiva of healthy young adult rabbits onto Millicell-CM Biopore filter units. After fixation with buffered glutaraldehyde and Giemsa staining, two × 200 images were selected from each sample representative of either slight multilayering or substantial multilayering, projected at × 1000, an overlay of the outlines of the goblet cells was made, and their dimensions and areas were measured. RESULTS: From measures of 4918 goblet cells, the average value (+/- SD) for the longest dimension was 17.7 ± 6.4 µm and 14.6 ± 5.3 µm for the shortest dimension. The GCD values ranged from 210 to 2069/mm2, with a mean of 1074 ± 601/mm2, but was lower for slightly multilayered images (at 537 ± 239 cells/mm ) compared with multilayered regions (at 1612 ± 601 cells/mm2; p < 0.001). The measured areas ranged from 72 to 491 µm2, with average values from any particular image ranging from 110 to 370 µm2, which were inversely correlated with the estimated GCD (Spearman's rho = - 0.722, p < 0.05). CONCLUSIONS: Larger goblet cells but in fewer numbers were predictably found across the filter surface where there were fewer layers of cells and vice versa. This difference could be considered in selection of images for counts of goblet cells from CIC specimens.

Changes in conjunctival epithelial cells after treatment with 0.2% xanthan gum eye drops in mild-moderate dry eye.

PURPOSE: To study the effects of xanthan gum eye drops on the ocular surface and conjunctival cytology of patients with mild-moderate dry eye. METHODS: This prospective, double-masked, controlled trial included 30 patients (age > 60 and Ocular Surface Disease Index score >12 and <33), divided into two groups of 15 subjects and treated with 0.2% xanthan gum eye drops (group 1) or 0.5% carboxymethylcellulose (group 2) qid. After a run-in period with saline qid, patients were evaluated by Ocular Surface Disease Index questionnaire, clinical assessment, and impression cytology at baseline (T0) and after 1 month (T1). For impression cytology, cellularity, cell-to-cell contacts, nucleus/cytoplasm ratio, chromatin aspect, goblet cells distribution, keratinization, and the presence of inflammatory cells were considered. Parameters were scored from 0 (no alterations) to 3 (evident alterations). For statistical analysis, Student's t-test, Wilcoxon rank-sum test, and Mann-Whitney U-test were used. RESULTS: Clinically, after 1 month of treatment, group 1 showed an improvement of corneal stain (T0 = 1.1 ± 1.4; T1 = 0.5 ± 0.7; p = 0.03) and a reduction of Schirmer I test (T0 = 9.8 ± 6.1; T1 = 5.9 ± 4.1; p = 0.001). In group 2, no differences were found between T0 and T1 for all the clinical tests. For impression cytology, in group 1 cellularity (T0 = 0.6 ± 0.5; T1 = 0.3 ± 0.5; p = 0.05), chromatin aspect (T0 = 1.2 ± 0.4; T1 = 0.8 ± 0.5; p = 0.01), keratinization (T0 = 1 ± 0.7; T1 = 0.5 ± 0.5; p = 0.03), and total score (T0 = 5.8 ± 1.3; T1 = 3.6 ± 1.7; p = 0.003) were significantly ameliorated, while in group 2 only total score improved significantly (T0 = 5 ± 1.4; T1 = 4.3 ± 1.5; p = 0.01). The comparison between groups showed significant amelioration for keratinization in group 1 at T1 (p = 0.02). CONCLUSION: The treatment with xanthan gum, a molecule with anti-oxidant and mucoadhesive properties, ameliorated conjunctival epithelium of mild-moderate dry eye patients better than carboxymethylcellulose.

Evaluation of IL8 pathway on the ocular surface: new insights in patients with ocular mucous membrane pemphigoid.

PURPOSE: To evaluate the expression of IL8/CXCL8 cytokine and its receptor CXCR1 in tear film and ocular surface of patients with ocular mucous membrane pemphigoid (oMMP). METHODS: Ten patients with oMMP in the quiescent phase, 20 patients with primary Sjogren syndrome (pSS) and 13 age- and sex-matched healthy controls (HCs) were included in this study. All patients undergone complete eye examination including lacrimal function tests and ocular surface staining assessed by ocular staining score. Ocular mucous membrane pemphigoid (oMMP) staging according to Mondino classification and dry eye severity grade according to Dry Eye Workshop 2007 classification were recorded. Tear samples and conjunctival epithelium were collected. IL-8 tear concentration was measured by enzyme-linked immunosorbent assay, and conjunctival IL8 was analysed by Western blot; conjunctival expression of CXCR1 was evaluated by immunohistochemistry. Il-8 and CXCR1 expression in oMMP patients were compared with HCand pSS patients and correlated with ocular clinical findings. RESULTS: Tear levels of IL-8 were significantly increased in patients with oMMP (260.1 ± 70 pg/ml) when compared to both HCs (98.5 ± 71.35 pg/ml; p = 0.001) and patients with pSS (96.3 ± 87.5 pg/ml; p = 0.001). Conjunctival expression of IL8 and CXCR1 was significantly increased in oMMP patients when compared to both healthy subjects and pSS patients. CONCLUSION: The significant increase of tear and conjunctival IL8 and CXCR1 levels in patients with oMMP when compared to healthy subjects and patients with Sjogren syndrome suggests that changes of IL8 pathway are specific of oMMP and may represent a potential biomarker of the disease and/or therapeutic target.

Multiple count sampling of goblet cells in microscope high-power fields using conjunctival impression cytology.

BACKGROUND: Published studies indicate that assessments of goblet cell density using conjunctival impression cytology has provided very variable results, but the reasons for this are unclear. Systematic analyses of the sources of variability are required. METHODS: From 20 healthy young adults, conjunctival impression cytology specimens were obtained using a supported filter unit applied to the superior bulbar conjunctiva. The filters were stained with Giemsa and 10 non-overlapping, randomly selected high-power field images were obtained from each specimen and the numbers of goblet cells per high-power field counted. RESULTS: From all 200 high-power fields assessed, the numbers of goblet cells ranged from zero to 74, with an overall mean value of 11.6 ± 14.8 per high-power field. From each successive set of 10 microscope field images from all individuals, the average number of goblet cells ranged from 23.2 in the first high-power field that obviously included numerous goblet cells down to 6.2 per high-power field. As the outcome from multiple counts/individual was systematically increased, these averages progressively decreased from 23.2 to 11.6 per high-power field, and while the standard deviation values also progressively declined (from 7.9 to 5.5 per high-power field), the relative variability (as the co-efficient of variation) did not, and increased to averaged values of over 100 per cent. CONCLUSIONS: These analyses indicate that there is a benefit of making multiple counts of goblet cells from different high-power fields, but that there is no obvious benefit of using more than five to seven high-power fields for any particular specimen.

Gene expression analysis of conjunctival epithelium of patients with Stevens-Johnson syndrome in the chronic stage.

OBJECTIVE: To investigate the pathology underlying the ocular surface complications of patients with Stevens-Johnson syndrome (SJS) in the chronic stage. METHODS AND ANALYSIS: Using oligonucleotide microarrays, we performed comprehensive gene expression analysis of the conjunctival epithelium of patients with SJS in the chronic stage (n=3). The controls were patients with conjunctival chalasis (n=3). We confirmed the downregulation and upregulation of transcripts of interest by quantitative real-time PCR (RT-PCR) assay. The expression of ocular surface protein with significantly upregulated transcripts was assessed immunohistochemically. RESULTS: Compared with the controls, in the conjunctival epithelium of patients with SJS, 50 transcripts were downregulated by less than one-tenth (analysis of variance (ANOVA) p<0.05). Transcripts MUC7, PIGR, HEPACAM2, ADH1C and SMR3A were downregulated by less than one-fiftieth. 65 transcripts were upregulated more than 10- fold; the difference between patients with SJS and the controls was significant (ANOVA p<0.05). There were 14 transcripts that were upregulated more than 50-fold; they were SERPINB4, KRT1, KRTDAP, S100A7, SBSN, KLK6, SERPINB12, PNLIPRP3, CASP14, ODZ2, CA2, CRCT1, CWH43 and FLG. Quantitative RT-PCR of conjunctival epithelium samples from 11 patients with SJS and 26 controls showed that the gene expression of PIGR, HEPACAM2 and ADH1C was significantly downregulated while the gene expression of ODZ2 (teneurin-2) was significantly upregulated in patients with SJS. We document that teneurin-2 protein can be expressed in human conjunctival epithelium. CONCLUSION: Our results suggest that the downregulation of PIGR, HEPACAM2 and ADH1C and upregulation of teneurin-2 expression contribute to the pathology of the ocular surface in patients with SJS in the chronic stage.

Examination of Kynurenine Toxicity on Corneal and Conjunctival Epithelium: In vitro and in vivo Studies.

Kynurenine (KYN) is a metabolite of tryptophan, proposed for the treatment of corneal diseases. Our goal was to evaluate the effects of KYN on normal human corneal and conjunctival epithelial cells in vitro and the re-epithelization of corneal erosion in rabbits. In our study, we used corneal (10.014 pRSV-T) and conjunctival (HC0597) epithelium cell cultures. KYN was applied at a concentration range of 1-100 µM for 24 and 48 h. We examined the effects on cellular metabolism, viability, interleukin-1ß (IL-1ß), IL-6, IL-10 secretion, cytoskeleton organization and transwell migration ability. Following a bilateral corneal de-epithelialization, the rabbits received drops containing 1% KYN and a saline solution to the contralateral control eye, 5 times daily. Digital images were analyzed using the EPCO 2000 software. The metabolic activity of cells was slightly decreased by KYN in the corneal but not in the conjunctival epithelium. The viability of both epithelia was improved by KYN; it caused alterations in the secretion of IL-6 and IL-10 but not IL-1ß. It had no impact on both epithelia morphology and the organization of the cellular cytoskeleton. KYN stimulated the formation of pseudopodia projections in both epithelia in vitro, which may be important in terms of wound healing. However, there were no differences in the re-epithelization rate in vivo. At the tested concentrations, KYN was not toxic for the corneal and the conjunctival epithelium in vitro and did not affect corneal re-epithelization in rabbits in vivo. Our results suggest that KYN may be taken into consideration for the treatment of ocular disorders.

Effect of Topically Epithelial Sodium Channel Inhibitors Eyedrops on Tear Production in Rabbit with Scopolamine Induced Dry Eye / 中山大学学报(医学科学版)

@#【Objective】To test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel（ENaC） will increase ocular hydration and cure the rabbit′ s dry eye induced by scopolamine. 【Methods】In the experiment，24 New Zealand rabbits weighing about 2.0- 2.5 kg were divided into 3 groups：tested group（8 rabbits）， control group （8 rabbits），and treatment group （8 rabbits）. For the rabbits in the tested group，they were given subcutaneous injection of scopolamine 4 times a day for 12 consecutive days to induce the dry eye. For the rabbits in the control group，they accepted subcutaneous injection of saline solution. For the rabbits in the treatment group ，they were firstly given the same treatment as the rabbits in the tested group. Then，the local regions in their right eyes received the application of 100 mmol/L amiloride （a sodium channel inhibitor）. Meanwhile，the local regions in their left eyes received the application of equivalent saline solution. We detected ENAC α and ENAC γ subunits of epithelial sodium channel in conjunctival epithelium by fluorescence quantitative PCR and detected the opening of epithelial sodium channel by short- circuit current technology. Finally，we compared the ENaC α and ENaC γ gene expression，corneal fluorescein sodium staining and tear secretion among the 3 groups. Fluorescence quantitative PCR was used to detect ENaC α and ENaC γ subunits of epithelial sodium channel in conjunctival epithelium and short-circuit current was used to detect the opening of epithelial sodium channel. The ENaC α and ENaC γ gene expression，corneal fluorescein sodium staining and tear secretion （immerged length） were compared among 3 groups.【Results】 The results showed that the quantity of tear secretion was（17.00 ± 0.37）mm for the control group，（4.42 ± 1.34）mm for the tested group（P<0.001 vs Control，n=8） and（14.25 ± 0.54）mm for the treatment group（P>0.05 vs Control，n=8）. The results of short-circuit current detection showed that the sodium current was（5.72 ± 0.35）μA /cm2 in normal model and（12.24 ± 0.54）μA /cm2 in dry eye model（P<0.001）. After Amiloride treatment，the sodium current decreased to（4.00 ± 0.61）μA/cm2 （P>0.05） and there was no statistical difference compared with the normal group. According to the results of fluorescence quantitative PCR，the expression of ENaC α and ENaC γ subunits and IL- β in in dry eye model were up-regulated（P<0.01） compared with that in normal group. After topical amiloride application，there were no statistical differences in inflammatory cytokines IL- 1 β，ENaC α，and ENaC γ between the normal group and treatment group（P>0.05，n=8）. After treatment，tear secretion increased （P<0.001），and ocular surface staining improved significantly.【Conclusion】 Topical application of amiloride increase the quantity of preocular tears owing to inhibition of conjunctival sodium channels and could provide an effective new therapy for chronic dry eye.

Tcf7l2 localization of putative stem/progenitor cells in mouse conjunctiva.

Conjunctival integrity and preservation is indispensable for vision. The self-renewing capacity of conjunctival cells controls conjunctival homeostasis and regeneration; however, the source of conjunctival self-renewal and the underlying mechanism is currently unclear. Here, we characterize the biochemical phenotype and proliferative potential of conjunctival epithelial cells in adult mouse by detecting proliferation-related signatures and conducting clonal analysis. Further, we show that transcription factor 7-like 2 (T-cell-specific transcription factor 4), a DNA binding protein expressed in multiple types of adult stem cells, is highly correlated with proliferative signatures in basal conjunctival epithelia. Clonal studies demonstrated that Transcription factor 7-like 2 (Tcf7l2) was coexpressed with p63α and proliferating cell nuclear antigen (PCNA) in propagative colonies. Furthermore, Tcf7l2 was actively transcribed concurrently with conjunctival epithelial proliferation in vitro. Collectively, we suggest that Tcf7l2 may be involved in maintenance of stem/progenitor cells properties of conjunctival epithelial stem/progenitor cells, and with the fornix as the optimal site to isolate highly proliferative conjunctival epithelial cells in adult mice.

On the Variability in Goblet Cell Density in Human Bulbar Conjunctival Samples Collected by Impression Cytology with Millicell-CM Biopore Membrane Units.

PURPOSE: To assess whether or not the numbers of goblet cells were different across regions of a conjunctival impression cytology (CIC) specimen. METHODS: CIC specimens were obtained from the exposed nasal bulbar conjunctiva from 22 healthy young Caucasian adults, stained with Giemsa and evaluated at a final magnification of 200× (medium power fields). Up to 14 different non-overlapping microscope fields, depending on whether or not a field was without goblet cells, were randomly selected. RESULTS: A microscope field could contain between 0 and 145 goblet cells. If only fields that included goblet cells were analyzed, the mean goblet cell counts (22 specimens, 10 fields/specimen) ranged from 37.8 to 44.8/field (equivalent to 259-313 goblet cells/mm2). When 3, 5 or 10 fields were used to calculate average goblet cell counts for each specimen, the standard deviation values were between 15.8/field and 21.7/field, with the lower values obtained if 10 fields were used. The resultant averaged inter-sample variability, as the coefficient of variation (COV), ranged from 40.0% to 57.5%, while the averaged intra-sample variability in counts ranged from 52.1% to 73.9%. If fields without goblet cells were also used, the resultant mean goblet cell count (from 10 fields/specimen) was statistically lower at 33.1/field (232 goblet cells/mm2). CONCLUSIONS: These analyses confirm various subjective comments made by early CIC investigators; the distribution of goblet cells across a CIC filter may be highly variable.