Conjunctival Epithelium Research Articles

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (β-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Transmembrane Mucin Response in Conjunctival Epithelial Cells Exposed to Wall Shear Stresses

Human conjunctival epithelium cells (HCEC) line the inner surface of the eyelid and cover the sclera and are continuously subjected to wall shear stresses (WSS). The effects of external forces on the conjunctival epithelium are not fully known. The conjunctival epithelium contains stratified squamous cells that synthesize the membrane-spanning mucins MUC1 and MUC16, which play important roles in protecting the ocular surface. Alterations in both gel-forming and membrane-tethered mucins occur in drying ocular surface diseases. The aim of this study was to explore the mechanobiological characteristics of transmembrane mucin secretion and cellular alterations of primary HCEC exposed to airflow-induced WSS perturbations. We exposed the HCEC to a steady WSS of 0.5 dyne/cm2 for durations of 15 and 30 min. Cytoskeletal alterations and MUC1 secretions were studied using immunohistochemically fluorescent staining with specific antibodies. We investigated for the first time an in vitro model of membrane-tethered mucin secretion by HCEC in response to WSS. The exposure of HCEC to WSS increased the polymerization of F-actin, altered the cytoskeletal shape and reduced the secretion of membrane-tethered MUC1.

Unilateral conjunctival concretions associated with chronic inflammation and tear instability.

Conjunctival concretions are small yellow-white subconjunctival spots that can be asymptomatic for years and are an incidental finding on routine ophthalmic examination.[1,2] The etiologies are chronic vernal conjunctivitis, severe atopic keratoconjunctivitis, and post-trachomatous changes.[1–5] The concretions are located within the conjunctival recesses layered by epithelium and are made up of cell debris and phospholipids.[3,4] Case Report A 24-year-old-female presented with chronic irritation and redness in the right eye for 1 year. Systemic evaluation was unremarkable with no immunosuppressive disorders. Her symptoms were intermittently associated with discharge. Her vision was 20/20 in both eyes. Everted upper and lower eyelids of the right eye showed multiple concretions scattered beneath the tarsal conjunctiva, more in the upper eyelid, protruding through the conjunctiva at two focal points [Fig. 1]. There was diffuse congestion in the tarsal conjunctiva with a normal lid margin appearance. The left eyelid had neither congestion nor concretions. There was no ocular surface staining with fluorescein dye, but the exposed concretions showed fluorescein uptake. Dry eye workup revealed a tear break-up time of 7.8 s with normal tear secretion in the right eye, whereas tear breakup and secretion was normal in the left eye. She gave an antecedent history of a vegetative object falling into her right eye while driving a bike. The ocular symptoms developed 1 month after that trauma. She was using topical lubricants and antibiotic ointment over the eyelid margin for the meibomitis. Because of unilateral presentation and young age, two concretions were removed on slit lamp using a sterile 26-gauge needle under topical anesthesia and sent for tissue processing.Figure 1: (a) Everted right upper eyelid of the 24-year-old female shows scattered numerous concretions in the tarsal conjunctiva, fewer in the lower eyelid (d). (b) Gomori methenamine silver stain (100×) shows presence of fungal elements within the concretions (higher magnification in inset), which are also seen on gram stain (e, 400×) as empty round and vertical spaces. (c and f) Left upper and lower eyelids show normal tarsal conjunctivaThe histopathologic examination of concretions revealed eosinophilic homogenous amorphous material containing fungal elements, which were Gomori methenamine silver stain positive [Fig. 2]. The deepest sections (till the last tissue section) also contained numerous fungal hyphae, which ruled out contamination. Due to atypical histopathologic findings of concretions, a superficial conjunctival incision biopsy containing tarsal conjunctiva, concretions, and a thin sliver of tarsus was taken at 4 mm from the upper eyelid margin. The incision biopsy showed scattered amorphous, acellular eosinophilic hyaline-like material and diffuse lymphocytic infiltration in the substantia propria. The amorphous material had a rim of conjunctival epithelium suggestive of concretions embedded within the pseudoglands of Henle. The periphery of the concretions had a slight basophilic appearance. The concretions were negative for von Kossa stain, Alcian blue, and Congo red but were red on Masson trichrome stain, suggesting cell debris and keratin to be one of the constituents. Alcian blue was focally positive in the conjunctival epithelium at the location of goblet cells. She was started on tablet fluconazole 150 mg once a week for 3 weeks along with 1% itraconazole eye ointment into the eye twice a day. Her symptoms resolved with a reduction in the conjunctival concretions’ number and conjunctival congestion.Figure 2: (a) Everted right upper eyelid with highlighted biopsied part. (b and c) Incisional biopsy from the upper tarsal conjunctiva shows diffuse subepithelial lymphocytic infiltration along with eosinophilic hyaline-like material deposits (c, at 200×). Masson trichrome (d), Congo red (e), and Alcian blue (f) were negative for amyloid and mucopolysaccharide depositsTear film instability and reduced tear production have been reported in patients with conjunctival concretions, where 51% of asymptomatic patients with conjunctival concretions have reduced tear break-up time.[3] The current case had reduced tear break-up time in the eye with concretions. An interesting finding in the current case was the presence of fungal elements, which can be explained by the possible entrapment of organic body containing fungus 1 year back. The organic material dissolved away, but the fungal elements caused persistent chronic inflammation, which resulted in the formation of concretions. This case reports the possibility of having unilateral concretions secondary to chronic inflammation with degenerative changes. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Hyderabad Eye Research Foundation, Hyderabad. Conflicts of interest There are no conflicts of interest.

A new perspective on the corneo‐scleral junction with three types of microscopy techniques

The conjunctions of the cornea and sclera in the eyes of donkeys, cattle, dogs, sheep, pigs and rabbits, regardless of gender, were examined in this study. No animals were specifically sacrificed for this investigation. Scanning electron microscopy, light microscopy, and dissecting microscopy were used in this research. In the limbus of all the animals investigated, the cornea and sclera fused in accordance with a pattern. At the corneo-scleral junction, the sclera was situated anteriorly and the cornea posteriorly in the dorsal and ventral sections of the bulbus oculi. In the medial and lateral parts of the eyeball, the cornea and sclera were facing each other and interlaced. Pigmentation and the sulcus scleralis externus could be used to identify the macro-and micro-anatomical boundaries of the limbus. In addition, the cytoplasm of basal epithelial cells shrank, signaling the end of the corneal epithelium and the start of the conjunctival epithelium. The presence of Bowman's membrane in cattle and sheep eyes was definitely determined in histological examinations. Bowman's membrane in these animals came to an end at the limbus, which is where the conjunctival epithelium starts and the corneal epithelium ends. In all areas of the cornea, Bowman's membrane revealed irregular, abrupt thickening and thinning. The corneal epithelium was thick in the vertex and thinner towards the limbus, whereas Descemet's membrane was thin in the center (vertex) and thick in the periphery (near the limbus). In this study, pictures and diagrams were used to illustrate the general anatomical, histological, and morphometric characteristics of the limbus in the species under investigation. The data from our study showed that the limbus region of the bulbus oculi was narrow in the lateral and medial parts and wide in the dorsal and ventral parts. This was confirmed in the studied animals as a general rule. The width value will undoubtedly affect the number of cells covered by the regions. It is conceivable that these cells will play a significant role in the decision of where to perform surgical procedures in order to promote wound healing, giving doctors an advantage. In this circumstances, we think that the limbus should be studied in terms of clinical methods because it has different shapes depending on the species and the position of the bulbus.

Effect of immobilized hyaluronidase and subtilisin enzymes on the ultrastructure of human conjunctival epithelial cells

The use of enzyme preparations is a traditional trend in various fields of medicine. The usage of immobilized enzymes hyaluronidase (IG) and subtilisin (IS) seems to be very promising for the treatment of damage to the ocular surface. The aim of the study was to establish the nature of changes in the ultrastructural organization of human conjunctival epithelium under the influence of immobilized hyaluronidase and subtilisin in vitro. Material and methods. In the experiment, a culture of normal cells of the human conjunctiva Chanqconjunctiva, clone 1–5 C-4, was seeded in 96well plates in the amount of 2 × 104 cells/well, after 24 h the medium was removed, Eagle’s medium MEM and fetal calf serum were added and cells were cultured for another 48 h. 5 experimental groups were formed: group 1 – without drugs (except for the composition of the incubation scheme), groups 2 and 3 – with IS at a concentration of 37 and 150 U/ml, respectively, groups 4 and 5 – with IG at a concentration of 37 and 150 U/ml, respectively. After preparation of cell preparations under a JEM 1400 electron microscope (Japan), ultrathin sections of 70–100 nm were studied at ×1000 magnification. Results. The article presents the results of electron microscopy of a culture of normal cells of the human conjunctiva in 5 experimental groups of cells with a description of changes in cytoarchitectonics under the influence of IG and IS. Conclusions. The introduction of immobilized enzymes into human conjunctival cell culture at a low dose (37 U/ml) affects the organization of cells without cytotoxic effects, while increasing the dose directly correlates with the occurrence of a cytotoxic effect.

Limbal Stem Cell Deficiency: A Review Focusing on Staging, Diagnostic and Treatment Modalities

The cornea being a transparent tissue acts as a refractive surface as well as a protective barrier of the eye. The corneal epithelium is being continuously replaced and renewed by the Limbal Stem Cells (LSC). The turnover of corneal epithelium is brought about by asymmetrical differentiation and self-renewal of LSCs, their migration to the cornea and desquamation of current corneal epithelium. LSC are undifferentiated cells that acts as progenitors of corneal epithelium and maintains corneal homeostasis. LSC resides in its own microenvironment known as LSC niche, which are capable of sustaining these tissue regenerating cells. Any intrinsic factors (e.g., toxic epidermal necrolysis or mucous membrane pemphigoid), extrinsic factors (e.g., thermal burns, radiations, chemical burns) or genetic defects (e.g., aniridia) cause impairment of LSC or its niche leading to Limbal Stem Cell Deficiency (LSCD). LSCD is associated with invasion of cornea by conjunctival epithelium, corneal opacity, and visual impairment. The diagnostic modality of LSCD includes fluorescein staining and slit lamp examination, impression cytology, confocal scanning and anterior chamber optical coherence tomography. The staging of LSCD is important for deciding the treatment modalities. In LSCD, the most common treatment modality includes LSC transplant from a healthy eye. Non LSC transplantation techniques are being used to prevent allograft rejection. In this review article, the authors aim to summarise the existing knowledge of the aetiology, staging and treatment modalities of LSCD.

Impression Cytology in the Evaluation of Ocular Surface Squamous Neoplasia- A Retrospective Cohort Study

Introduction: Ocular Surface Squamous Neoplasia (OSSN) are a diverse group of lesions varying from Conjunctival Intraepithelial Neoplasia (CIN) to invasive Squamous Cell Carcinomas (SCC). Histopathological examination is the gold standard technique in diagnosing them. However, it is invasive and may not be suitable in recurrent cases. Numerous minimally invasive techniques are available to diagnose OSSN cytologically. This study employs Impression Cytology (IC) to diagnose ocular squamous neoplastic lesions and compare it with the histological diagnosis. Aim: To utilise IC in the diagnosis of OSSN and to compare its findings with histopathological diagnosis. Materials and Methods: This was a retrospective cohort study, conducted in a Tertiary Eye care Hospital, Hubli, Karnataka, India during January 2023. The data included was over a period of three years (from July 2016 to June 2019). A total of 44 eyes (34 patients), in which both biopsy and IC done, were included. The patients’ age, sex and ocular examination findings were recorded. The cytological and histopathological slides were retrieved from the archives and examined morphologically. The cytological findings were compared with histopathological diagnosis and the sensitivity and specificity of the IC technique were calculated. The data was analysed using Microsoft excel sheets. Results: A total of 34 patients were studied ranging from 19 to 85 years. The mean age was 49 years, 21 (61.76%) of them were males and 13 (38.24%) were females. In 24 patients, single eye was affected and in 10 patients bilateral eye involvement was seen. Histology revealed that there were 18 (40.90%) CIN I cases, 4 (9.09%) CIN II, 5 (11.36%) CIN III and 17 (38.65%) SCC. On cytology, 3 (6.82 %) were normal conjunctival epithelium, 30 (68.18 %) were dysplasia and 11 (25.00 %) were SCC. On cyto- histomorphological analysis, 35 (79.55 %) were concordant, out of 44 diagnosis. IC has a sensitivity of 88.89% and specificity of 64.71% to detect dysplasia. To diagnose SCC, it has sensitivity of 64.71% and 100% specificity. Conclusion: According to the present study observations, IC is an excellent preliminary tool to investigate suspected neoplastic lesions of ocular surface. It has a very good concordance with histopathological diagnosis. IC is highly sensitive to detect dysplasia (CIN) and is very specific to diagnose SCC.

High dupilumab levels in tear fluid of atopic dermatitis patients with moderate-to-severe ocular surface disease.

The patho-mechanism of ocular surface disease (OSD) in dupilumab-treated atopic dermatitis (AD) patients remains unclear. The aim of this study is to measure dupilumab levels in tear fluid and serum, and relate these findings to the severity of OSD during dupilumab treatment in AD patients. This prospective study included dupilumab-treated moderate-to-severe AD patients who were seen by a dermatologist and an ophthalmologist before the start of dupilumab (baseline), and after 4 and 28weeks of dupilumab treatment. Dupilumab levels in tear fluid and serum were measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Additionally, a pilot study was conducted to measure dupilumab on conjunctival epithelial cells using flow cytometry and LC-MS/MS. At baseline, 89.6% (n=43/48) of the patients had OSD, with 50.0% having moderate-to-severe OSD. After 28weeks of dupilumab treatment, the median dupilumab tear fluid levels were 0.55mg/L (IQR 0.35-1.31) and 0.29mg/L (IQR 0.16-0.60) in patients with moderate-to-severe OSD and patients with no or mild OSD, respectively (p=0.02). Dupilumab levels could be detected on conjunctival epithelial cells of 5 AD patients treated with dupilumab for 4weeks. Patients with moderate-to-severe OSD had higher dupilumab tear fluid levels compared to patients with no or mild OSD, indicating that dupilumab reaches the ocular surface. Dupilumab was also detected in conjunctival cell suspensions and was found to directly bind CD45-conjunctival epithelial cells. This suggests that AD-induced changes of the conjunctival epithelium may play a role in the development of OSD as well as increased local drug availability.

In vitro infection of human ocular tissues by SARS-CoV-2 lineage A isolates

BackgroundThe purpose of this study was: [1] to evaluate the infectivity of two SARS-CoV-2 lineage A variants on human ocular tissues in vitro, and [2] to evaluate the stability of SARS-CoV-2 lineage A variants in corneal preservation medium.MethodsPrimary cultures of donor corneal, conjunctival, and limbal epithelium were inoculated with two lineage A, GISAID clade S isolates of SARS-CoV-2 (Hong Kong/VM20001061/2020, USA-WA1/2020), to evaluate the susceptibility of the ocular tissue to infection. Flat-mounted Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) grafts were inoculated with SARS-CoV-2 to evaluate the susceptibility of the endothelium to infection. All inoculated samples were immunostained for SARS-CoV-2 nucleocapsid (N)-protein expression to confirm positive infection. SARS-CoV-2 Hong Kong was then inoculated into cornea preservation media (Life4°C, Numedis, Inc.). Inoculated media was stored at 4oC for 14 days and assayed over time for changes in infectious viral titers.ResultsCorneal, conjunctival, and limbal epithelial cells all demonstrated susceptibility to infection by SARS-CoV-2 lineage A variants. Conjunctiva demonstrated the highest infection rate (78% of samples infected [14/18]); however, infection rates did not differ statistically between cell types and viral isolates. After inoculation, 40% (4/10) of DSAEK grafts had active infection in the endothelium. SARS-CoV-2 lineage A demonstrated < 1 log decline in viral titers out to 14 days in corneal preservation media.ConclusionsSARS-CoV-2 lineage A variants can infect corneal, limbal, and conjunctival epithelium, as well as corneal endothelium. There was no statistical difference in infectivity between different lineage A variants. SARS-CoV-2 lineage A can survive and remain infectious in corneal preservation media out to 14 days in cold storage.

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.

Anterior segment optical coherence tomography in ocular surface tumours and simulating lesions.

This study aims to systematically review the reported literature on the use of anterior segment optical coherence tomography (AS-OCT) in ocular surface tumours and simulating lesions. A systematic literature search was done using PubMed, Scopus, and Web of Science databases between January 2002 and December 2021. On AS-OCT, ocular surface squamous neoplasia typically demonstrate epithelial thickening, epithelial hyperreflectivity, and an abrupt transition between normal and abnormal epithelium. Conjunctival nevi usually show mildly hyperreflective epithelium of normal thickness, internal hyperreflectivity, and intralesional cysts which is the hallmark of this tumour. Primary acquired melanosis presents with normal thickness epithelium, basal epithelial hyperreflectivity, and absence of cysts. Conjunctival melanoma demonstrates hyperreflective normal/thickened epithelium, hyperreflective basal epithelium, internal hyperreflectivity, and absence of intralesional cysts. Conjunctival lymphoma shows homogenous, low-medium reflective subepithelial lesions with smooth borders, and dot-like infiltrates. Benign reactive lymphoid hyperplasia findings are similar to lymphoma but the infiltrates are more hyperreflective compared to lymphoma. Pterygium shows thickened conjunctival epithelium, epithelial hyperreflectivity, and subepithelial wedge-shaped hyperreflective tissue separated from the overlying epithelium by a cleavage plane. Pinguecula demonstrates mildly thickened epithelium and similar findings with pterygium but does not extend beyond the corneal limbus. This review shows that AS-OCT, as a noninvasive tool, has potential uses in the differential diagnosis of ocular surface tumoursand simulating lesions. Major limitations of AS-OCT include limited visualization of the posterior border of thick, keratinized, and pigmented tumours and lack of assessment of large conjunctival tumours in a single cut.

Expression of GPR-68 in Human Corneal and Conjunctival Epithelium. Possible indicator and mediator of attrition associated inflammation at the ocular surface

Attrition and osmotic stress have been identified as major forces in the pathophysiology of dry eye. Impaired tolerance to mechano-transduction in the presence of insufficient lubrication has been associated with disturbances of ocular surface homeostasis and encouragement of inflammatory reactions, challenging the usual regulatory coping mechanisms. In spite of the probable link between enhanced attrition and secondary inflammation, the key mediators driving the vicious cycle of severe dry eye disease have not yet been identified. The goal of this study was therefore to investigate human corneal and conjunctival epithelium for the presence of the G protein-coupled receptor GPR-68. This protein had most recently been shown to be not only chemically activated but also mechanically, possibly through attrition. De-identified sections of human cornea and conjunctiva were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemical methods. Results Specific staining for G-protein-coupledreceptor 68 (GPR68) was observed in all samples of the cornea throughout the epithelial layers of the corneal epithelium, most prominently in the area of the wing cells and the basement membrane. Even in the conjunctiva, specific staining for GPR-68 was found. The detection of G protein-coupled receptor GPR-68 in human corneal and conjunctival epithelium raises the question of its function and purpose. The mechanical activation of GPR68 in situations with enhanced friction and attrition could modify various cellular functions and possibly jeopardize normal inflammatory homeostasis at the ocular surface. Accordingly, decreased lubrication in dry eye disease could result in activation of GPR-68. This could lead to secondary inflammation, initially in the epithelium and surrounding stroma. Continuous mechanical stress could result in chronic inflammation, also reaching deeper structures of the cornea, possibly making GPR-68 an important actor in the vicious cycle of dry eye disease. G protein-coupled receptor GPR-68, sensitive to flow and mechanic stimulation, is present in the human corneal epithelium and conjunctiva. Decreased lubrication and increased attrition, accompanied by sensations typical for dry eye, might lead to local inflammation. It is possible that subtle signs of conjunctival, and later corneal, surface damage in the context of these sensations could be a better indicator of the need for and success of therapy than the clinical signs of dry eye disease alone, at least in the early stages of the disease. Inhibition of G protein-coupled receptor GPR-68 could represent a new strategy in the treatment of dry eye disease.

Ex vivo models of corneal epithelial regeneration in bioreactor: Respective roles of limbal, conjunctival and corneal epithelia

AbstractPurpose: The corneas preserved in bioreactor (BR) had been shown to have not only a better endothelial viability, but also a more differentiated and stratified epithelium than corneas preserved in organoculture. Purpose: By using BR, we would analyse the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration.Methods: Five pairs of corneas from body donation to Science were used with a death‐to‐collection time &lt; 20 h. A 3‐ to 5‐mm‐wide conjunctival flange was kept intact. Five patterns were set up by fully mechanical removal of 1, 2, or 3 epithelia: C‐L + Conj+, C‐L‐Conj+, C‐L + Conj‐, C + L‐Conj‐, C‐L‐Conj‐ (control) n = 2 for each pattern. The limbus was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21 mmHg, 2.5 μl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analysed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer.Results: No epithelium was reconstituted in the controls. In the other 4 models including the C‐L‐Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression.Conclusions: In this BR model, conjunctival epithelial cells allowed the regeneration of a typical corneal epithelium in model C‐L‐Conj+. The corneal epithelium can also migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia (including the conj) contain stem cells or progenitors capable of migrating throughout the cornea and restoring the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti‐angiogenic barrier rather than the loss of limbic stem cells.

In vitro evaluation of sodium hyaluronate protective effect against benzalkonium chloride toxicity

AbstractPurpose: To evaluate the protective effect of different concentrations of sodium hyaluronate (SH) on the Benzalkonium chloride (BAK)‐induced toxicity using an in vitro model.Methods: The NAV14 cell line (SV40‐immortalized murine conjunctival epithelium) was used. Cell monolayers were exposed to different combinations of BAK (0.001%; 0.005%; 0.01%) and SH (0.2%; 0.3%; 0.4%) for 15 minutes; then cells were washed, and fresh culture media was added. Cell viability was evaluated after 2 h by resazurin reduction and lactate dehydrogenase (LDH) release. Also, cell migration and proliferation over 24 hours were determined by the scratch wound‐healing assay. Data were analysed by two‐way ANOVA and are shown as mean ± SD of two independent experiment with 4–6 replicates each.Results: BAK induced a concentration‐dependent decrease on cell viability (BAK 0.001%: 91 ± 14%, BAK 0.005%: 45 ± 9% and BAK 0.01%: 22 ± 10% of control cells, p &lt; 0.001) and an increase in LDH release (no BAK:0.22 ± 0.02, BAK 0.001%: 0.31 ± 0.02, BAK 0.005%: 1.14 ± 0.05 and BAK 0.01%: 1.21 ± 0.05, p &lt; 0.001). Conversely, SH neutralized these effects also in a concentration‐dependent manner (p &lt; 0.001). In the presence of SH 0.4% (highest effect), cell viability was BAK 0.001%: 104 ± 22%, BAK 0.005%: 109 ± 9% and BAK 0.01%: 75 ± 13% of control cells (p &lt; 0.001 for BAK 0.005–0.01%) while LDH release was no BAK: 0.24 ± 0.03, BAK 0.001%: 0.26 ± 0.01, BAK 0.005%: 0.37 ± 0.02 and BAK 0.01%: 0.49 ± 0.22, (vs. no SH: p &lt; 0.001 for BAK 0.005–0.01%). BAK also reduced wound closure in vitro (after 24 h, no BAK: 76 ± 14%, BAK 0.001%: 42 ± 12%, BAK 0.005%: 17 ± 16% and BAK 0.01%: 0.0% wound closure, p &lt; 0.001 for all BAK). Conversely, SH neutralized this effect in a concentration‐dependent fashion (p &lt; 0.001). In the presence of SH 0.4% (highest effect), wound closure at 24 h was: no BAK: 81 ± 15%, BAK 0.001%: 58 ± 6%, BAK 0.005%: 63 ± 10%, BAK 0.01%: 60 ± 8% (vs no SH: p &lt; 0.001 for BAK 0.005–0.01%).Conclusions: Sodium hyaluronate neutralized BAK toxicity on conjunctival epithelial cells in a concentration‐dependent manner. SH 0.4% was even protective at the highest preservative concentrations. These findings support the use of SH to mitigate BAK toxicity in long‐term antiglaucomatous treatment, although more studies are needed.

The effect of amniotic membrane proteins released by ocular inserts containing self‐healing hydrogels

AbstractPurpose: Growth factors found in the amniotic membrane (AM) help accelerate epithelium regeneration in the damaged cornea or conjunctiva. This study compares the effects of hydrogels releasing the AM proteins on the proliferation, migration, and time‐to‐closure (healing) of the conjunctival epithelium.Methods: Proteins extracted from the AM using sonication were incorporated into two hydrogels used as ocular inserts. The tested inserts materials were the self‐healing, dynamic hyaluronic‐acid hydrogel based on gold‐thiolate/disulfide exchange (Hydrogel A) and a physically cross‐linked hydrogel based on a combination between Eudragit S100 and hyaluronic acid with a reinforced matrix by polyvinyl alcohol, polyvinyl pyrrolidone and polyethylene glycol (Hydrogel B). In an in vitro experiment, a scratch was made on the human conjunctival epithelium cells (Chang Conjunctiva, Clone 1‐5c‐4) to mimic an incision wound. The cells were cultured with hydrogels for 48 hours to study the effect of these materials on epithelium repair. The samples were examined using a Zeiss Observer Z1 inverted microscope, and the results were evaluated employing the Image‐J program to obtain the cell migration rate (μm/h).Results: Hydrogel A increased the rate of closure at 22 h (121 ± 19 μm) in comparison with Hydrogel B (394 ± 78 μm). After 46 h, the scratches in Hydrogel A and control cultures were completely closed, while the Hydrogel B samples had an unclosed area of 54 ± 4 μm.Conclusions: Both hydrogels promoted wound healing in cultured conjunctival epithelium cells. However, a lower rate of wound closure was observed for Hydrogel B than for Hydrogel A and the control, probably because the speed of cell proliferation was slower in Hydrogel B. The wound‐healing assay showed that the Hydrogel A and B increased cell viability and significantly stimulated cell migration.

Long-Term Culture of Canine Ocular Cells That Maintain Canine Papillomaviruses.

Canine ocular papillomas occur on the haired skin of eyelids, conjunctival epithelium, and rarely on the cornea. Using PCR typing assays with canine papillomavirus type-specific primer sets, our study confirmed that the papillomas contained canine papillomavirus type 1. The positive result from a rolling circle amplification assay indicated the CPV1 viral genome in the cells is a circular episomal form. We also successfully established the first canine corneal cell line using the conditional reprogramming method. The cells exhibited an epithelial cell morphology, grew rapidly in vitro, and could be maintained long term. For the continued growth of the canine corneal cells, feeder cells played a more important role than Rho-kinase inhibitor Y-27632. More importantly, the viral CPV1 genome was maintained in the canine corneal cells during the long-term expansion. Unlimited supplies of these cells provide as a model for the study CPV in dog cells, and a platform for drug screening for effective therapies against canine papillomavirus infection in the future.

Conjunctival Lymphangiectasia Simulating Loa Loa.

A 46-year-old woman presented with complaints of irritation in the OS for 1 year. Her ocular examination revealed normal vision and anterior segment except for an incidental finding of a worm-like lesion in the temporal conjunctiva. The lesion was transparent, linear in shape forming a complete loop, extending from the superotemporal conjunctiva to the inferior conjunctiva (Panel A). The plane of the lesion was conjunctival. The lesion showed no mobility with exposure to light or when touched with a cotton-tip applicator. Differential diagnosis of dead loa loa versus lymphangiectasia was made. Her peripheral blood smear did not reveal any microfilariae and had a normal cell count. Surgical excision revealed the lesion to be embedded within the conjunctiva and no worm came out. Only a segment of the lesion was sent for biopsy as there was no worm. Histopathology revealed hyperplastic conjunctival epithelium (Panel B; ×200) showing diffuse and intense expression with pan-cytokeratin antibody (Panel C; ×200). Subepithelial stroma showed thickened, dilated, and ectatic vessels (Panel D; ×400). The endothelial lining of vessels showed strong and intense expression with CD34 and podoplanin antibodies (Panels E and F; ×400) indicating lymphatic origin. The final diagnosis of conjunctival lymphangiectasia was made. Isolated conjunctival lymphangiectasia can be an incidental finding or associated with chemosis or persistent irritation. A trial of topical steroids can be given in symptomatic patients. Surgical excision with or without amniotic membrane graft or conjunctival autograft can be performed in nonresponsive cases. Anterior segment optical coherence tomography of conjunctival lymphangiectasia shows multiple subepithelial cystic spaces within the conjunctiva.

Specific decellularized extracellular matrix promotes the plasticity of human ocular surface epithelial cells.

The ocular surface is composed of two phenotypically and functionally different epithelial cell types: corneal and the conjunctival epithelium. Upon injury or disease, ocular surface homeostasis is impaired resulting in migration of conjunctival epithelium on to the corneal surface. This can lead to incomplete transdifferentiation toward corneal epithelial-like cells in response to corneal basement membrane cues. We show that corneal extracellular matrix (ECM) proteins induce conjunctival epithelial cells to express corneal associated markers losing their conjunctival associated phenotype at both, mRNA and protein level. Corneal epithelial cells behave the same in the presence of conjunctival ECM proteins, expressing markers associated with conjunctival epithelium. This process of differentiation is accompanied by an intermediate step of cell de-differentiation as an up-regulation in the expression of epithelial stem cell markers is observed. In addition, analysis of ECM proteins by laminin screening assays showed that epithelial cell response is laminin-type dependent, and cells cultured on laminin-511 showed lower levels of lineage commitment. The phosphorylation and proteolysis levels of proteins mainly involved in cell growth and differentiation showed lower modifications in cells with lower lineage commitment. These observations showed that the ECM proteins may serve as tools to induce cell differentiation, which may have potential applications for the treatment of ocular surface injuries.

Identification of the circRNA-miRNA-mRNA Regulatory Network in Pterygium-Associated Conjunctival Epithelium.

To investigate the regulatory mechanism of pterygium formation, we detected differentially expressed messenger RNAs (DE-mRNAs) and differentially expressed circular RNAs (DE-circRNAs) in pterygium-associated conjunctival epithelium (PCE) and normal conjunctival epithelium (NCE). Genome-wide mRNA and circRNA expression profiles of PCE and NCE were determined using high-throughput sequencing. Bioinformatics analyses, including Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) analysis, were conducted. The microRNAs (miRNAs) interacting with the hub DE-mRNAs and DE-circRNAs were predicted and verified using real-time quantitative PCR (RT-qPCR). The data showed that there were 536 DE-mRNAs (280 upregulated and 256 downregulated mRNAs) and 78 DE-circRNAs (20 upregulated and 58 downregulated circRNAs) in PCE. KEGG enrichment analysis indicated that the DE-mRNAs were mainly involved in the following biological processes: IL-17 signalling pathway, viral protein interaction with cytokine and cytokine receptor, cytokine-cytokine receptor interaction, ECM-receptor interaction, and focal adhesion. The GSEA results revealed that the epithelial mesenchymal transition (EMT) process was significantly enriched in upregulated mRNAs. The pterygium-associated circRNA-miRNA-mRNA network was established based on the top 10 DE-circRNAs, 4 validated miRNAs (upregulated miR-376a-5p and miR-208a-5p,downregulated miR-203a-3p and miR-200b-3p), and 31 DE-mRNAs. We found that miR-200b-3p, as a regulator of FN1, SDC2, and MEX3D, could be regulated by 5 upregulated circRNAs. In addition, we screened out EMT-related DE-mRNAs, including 6 upregulated DE-mRNAs and 6 downregulated DE-mRNAs. The EMT-related circRNA-miRNA-mRNA network was established with the top 10 circRNAs, 8 validated miRNAs (upregulated miR-17-5p, miR-181a-5p, and miR-106a-5p, downregulated miR-124-3p, miR-9-5p, miR-130b-5p, miR-1-3p, and miR-26b-5P), and 12 EMT-related DE-mRNAs. We found that hsa_circ_0002406 might upregulate FN1 and ADAM12 by sponging miR-26b-5p and miR-1-3p, respectively, thus promoting EMT in pterygium. Briefly, the study provides a novel viewpoint on the molecular pathological mechanisms in pterygium formation. CircRNA-miRNA-mRNA regulatory networks participate in the pathogenesis of pterygium and might become promising targets for pterygium prevention and treatment.

IL-33-induced keratoconjunctivitis is mediated by group 2 innate lymphoid cells in mice

BackgroundInterleukin-33 (IL-33) is involved in type 2 innate immunity by inducing type 2 cytokines, such as IL-5 and IL-13, through the activation of group 2 innate lymphoid cells (ILC2s) or T helper 2 (Th2) cells. We previously reported that mice overexpressing IL-33 (IL-33Tg) in the cornea and conjunctiva spontaneously develop atopic keratoconjunctivitis-like inflammation. Despite previous studies, it is not fully understood what types of immune cells contribute to the disease process of IL-33-induced keratoconjunctivitis. MethodsTo defect Th2 cells, IL-33Tg mice were crossed with Rag2KO mice. To defect ILC2s, IL-33Tg mice received bone marrow transplantations from B6.C3(Cg)-Rorasg/J mice that lacked ILC2. Immunostaining techniques were used to determine where ILC2 is distributed in the cornea and conjunctiva. We analyzed the transcriptomes of ILC2 from the conjunctiva by using single-cell RNA-seq analysis. To investigate whether tacrolimus reduces type 2 cytokine production by ILC2, ILC2 was cultured with tacrolimus, and the percentage of cytokine-producing ILC2 was examined. To investigate whether tacrolimus can inhibit IL-33-induced keratoconjunctivitis in vivo, IL-33Tg mice were treated with tacrolimus eye drops. ResultsILC2 infiltrated the conjunctival epithelium and subepithelial tissue. Keratoconjunctivitis developed spontaneously in Rag2KO/IL-33Tg mice, but keratoconjunctivitis was abolished in IL-33Tg mice lacking ILC2. ILC2 was not a uniform cluster but a heterogeneous cluster. Tacrolimus inhibited cytokine production from ILC2s in vitro, and tacrolimus eye drops inhibited keratoconjunctivitis in IL-33Tg mice in vivo. ConclusionsILC2 plays a pivotal role in IL-33-induced keratoconjunctivitis in mice.