Experimental interventions attenuate a conjunctival epidermal metaplasia model.

The conjunctiva is a non-keratinized, stratified columnar epithelium with characteristics different from the cornea and eyelid epidermis. From development to adulthood, a distinguishing feature of ocular versus epidermal epithelia is the expression of the master regulator PAX6. A conditionally immortalized conjunctival epithelial cell line (iHCjEC) devoid of stromal or immune cells established in our laboratory spontaneously manifested epidermal metaplasia and upregulated expression of the keratinization-related genes SPRR1A/B and the epidermal cytokeratins KRT1 and KRT10 at the expense of the conjunctival trait. In addition, iHCjEC indicated a significant decrease in PAX6 expression. Dry eye syndrome (DES) and severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome, cause the keratinization of the entire ocular surface epithelia. We used iHCjECs as a conjunctiva epidermal metaplasia model to test PAX6, serum, and glucocorticoid interventions. Reintroducing PAX6 to iHCjECs resulted in upregulating genes related to cell adhesion and tight junctions, including MIR200CHG and CLDN1. The administration of glucocorticoids or serum resulted in the downregulation of epidermal genes (DSG1, SPRR1A/B, and KRT1) and partially corrected epidermal metaplasia. Our results using an isolated conjunctival epidermal metaplasia model point toward the possibility of rationally "repurposing" clinical interventions, such as glucocorticoid, serum, or PAX6 administration, for treating epidermal metaplasia of the conjunctiva.

Phospholipid analyses of rabbit ocular surface tissues.

The tissues of the integument covering the ocular surface comprise a mucus membrane functioning as a protective physical barrier and has the ability to mount a defensive inflammatory response. Since lipid metabolism has a role in both of these functions, we studied normal membrane phospholipids (PL) of the cornea and bulbar conjunctiva to (1) determine baseline PL profiles of these tissues, (2) compare and contrast these individual PL metabolite profiles as well as groups of metabolites, and (3) describe pathway-specific metabolic interrelations among these tissues. Corneal and conjunctival tissue samples were isolated from rabbit eyes (n = 30) and extracted with chloroform-methanol using a modified Folch procedure. 31P nuclear magnetic resonance spectroscopy was used to qualitatively and quantitatively measure tissue PL profiles. The cornea and conjunctiva, respectively, have the following PL composition (mole % of total detected phospholipid): phosphatidylglycerol (PG) -, 0.4; lysophosphatidylethanolamine 1.2, -; phosphatidic acid -, 0.4; diPG (cardiolipin) 2.1, 3.5; unknown PL at the chemical shift of 0.13 δ 1.5, 0.9; ethanolamine plasmalogen 11.2, 13.0; phosphatidylethanolamine 11.5, 12.8; phosphatidylserine 8.9, 10.1; sphingomyelin 10.2, 10.7; lysophosphatidylcholine 0.9, 1.4; phosphatidylinositol 5.3, 5.3; phosphatidylcholine (PC) plasmalogen or alkylacylPC 2.2, 1.9; PC 45.1, 40.0. In addition, 28 PL metabolic indices were calculated from these data, which permitted pathway-specific lipid analyses. This study (1) establishes PL profiles of the two ocular tissues of the integument that cover the surface of the eye, (2) compares and contrasts indices comprised of ratios and combinations of PL, and (3) describes pathway-specific metabolic interrelations among these tissues to serve as baselines for studies involving the distribution of tissue phospholipids.

The lateral fornix orbitotomy: a novel surgical corridor to lacrimal gland lesions.

PURPOSE: Current practice for diagnostic biopsy of lacrimal gland lesions entails an orbitotomy procedure via an upper eyelid crease or lateral canthotomy skin incision. We describe a novel surgical technique to address these lesions via the lateral conjunctival fornix. METHODS: Retrospective case series of all patients who underwent a lateral fornix orbitotomy procedure for incisional or excisional diagnostic biopsy of lacrimal gland lesions. The procedure involves a conjunctival incision in the lateral fornix remote from the openings of the lacrimal ductules, and an intraperiosteal surgical corridor to access the lacrimal gland. RESULTS: The study cohort included 16 patients (3 male, 13 female) with a mean age of 48.3 years (range, 24.0-78,9 years). The sampled lesions involved the orbital lobe in 14 patients, the palpebral lobe in 1 patient, and the entire gland in 1 patient. A histopathological diagnosis was obtained in all cases. Postoperatively, new moderate adduction deficit developed in one patient (6.3%) that recovered after adhesiolysis of the conjunctival scar. 3 patients (18.8%) experienced transient mild limitation of adduction or abduction. There was no new or worse ptosis or dry eye disease related to the surgery. The mean length of postoperative follow-up was 1.3 years (median 1.0 years, range, 0.6-4.7 years). CONCLUSION: The lateral fornix orbitotomy approach was successful in obtaining biopsy specimens of histopathological diagnostic value. It provides transconjunctival access to the lacrimal gland without damage to the excretory lacrimal ductules or displacement of the eyelid support system.

Expression of placental growth factor, neuropilin-1, and neuropilin-2 in primary pterygium tissue.

PURPOSE: The aim of this study was to evaluate the expression of placental growth factor (PLGF), neuropilin-1 (NP-1), and neuropilin-2 (NP-2) molecules in primary pterygium tissue compared with normal conjunctival tissue. METHODS: The records of 42 patients who underwent excision surgery with autografts for primary pterygium (pterygium group) and 20 patients who underwent conjunctival nevus excision surgery (control group) in the same period were reviewed retrospectively. The samples obtained from the pterygium tissues in the pterygium group and the clean conjunctival tissues adjacent to the nevus in the control group were collected from the archive. Immunohistochemical stains of the primary antibodies-1/100 diluted PLGF, NP-1, and NP-2 (Abcam Cambridge Science Park, UK)-were applied to all groups. Staining intensities and the percentage of positive cells in epithelial, endothelial, stromal, and inflammatory cells were analyzed by an experienced pathologist. RESULTS: The positivity rates of PLGF and NP-2 expression in epithelial, endothelial, stromal, and inflammatory cells were found to be higher in the pterygium group than in the control group (PLGF: p < 0.001, p < 0.001, p = 0.001, and p < 0.001, respectively; NP-2: p < 0.001 for all). Staining intensities for PLGF and NP-2 were higher in the pterygium group than in the control group (PLGF: p < 0.001, p < 0.001, p = 0.005, and p < 0.001, respectively; NP-2: p < 0.001, p < 0.001, p = 0.001, and p < 0.001, respectively). However, no significant differences were found in any cell type in terms of NP-1 expression positivity rates (p = 0.730, p = 0.121, p = 0.524, and p = 0.624, respectively) or staining intensity (p = 0.716, p = 0.147, p = 0.147, and p = 0.780, respectively). CONCLUSION: PLGF and NP-2 levels were found to be higher in pterygium tissue, while there was no difference in NP-1. These results indicate the possible roles of NP-2 and PLGF in primary pterygium.

Luminescence of favipiravir in skin appendages and sclera. A controlled study and literature review.

BACKGROUND/OBJECTIVES: Favipiravir is an antiviral agent, recently used for COVID-19 infections. Several reports associate favipiravir intake with Wood's lamp fluorescence of hair, nails, and sclera. The present study was designed to elucidate the positivity rates, and sites of favipiravir-related fluorescence and to unravel the site-specific changes in fluorescence positivity rates by a function of time past exposure. METHODS: The study population comprised 50 patients and 50 control individuals. All patients in the patient group had received a full dose of favipiravir for COVID-19 infection. Fifty volunteers served as the control group. Wood's lamp examination was performed in a completely darkened room, and the positivity rate, extent, pattern, and distribution of fluorescence were recorded. RESULTS: Wood's light revealed fluorescence of the fingernails, toenails, sclera, and hair in 35 (70%), 35 (70%), 22 (44%), and 8 (16%) patients, respectively. No control individual tested positive by Wood's lamp. Statistical analysis revealed significant differences between patient and control groups in terms of Wood's light luminescence in the fingernails (p = .000), toenails (p = .000), sclera (p = .000) and hair (p = .003). Although fingernail, toenail, and hair fluorescence positivity rates declined or ceased at or after 91 days of favipiravir exposure, ocular fluorescence positivity rates were prolonged up to 188 days. CONCLUSIONS: These findings confirm that favipiravir may produce fluorescence of nails, sclera, and hair, detectable by Wood's light starting from the initial month and peaking at second- and third months following exposure to the medication. Although nail and hair fluorescence tend to abate after 3 months, ocular fluorescence may persist even longer than 6 months after cessation of the medication.

Clinical analysis of 37 Chinese patients with ocular amyloidosis: a single center study.

OBJECTIVE: To examine the clinical characteristics, diagnosis and treatment, and prognosis of ocular amyloidosis in a Chinese population. METHODS: A retrospective case series study was conducted. The clinical data of 37 patients with ocular amyloidosis were collected and the clinical characteristics, diagnosis and treatment, and prognosis were summarized and analyzed. RESULTS: The 37 patients included 12 males and 25 females ranging in age from 22 to 75 years, with median age of 49 years. The clinical signs and symptoms included a conjunctival mass in 37 patients (100%), periorbital discomfort or pain in 29 patients (61.9%), ptosis in 18 patients (23.8%), exophthalmos or eyeball displacement in 3 patients (14.3%), restricted eye movement in 2 patients (9.52%), vision loss in 1 patient (4.76%), and diplopia in 1 patient (4.76%). A total of 29 patients had only conjunctival involvement and 8 patients had concomitant orbital and conjunctival involvement. The main treatment for patients with conjunctival involvement was surgical resection. Thirty-one patients had stable disease, 4 patients progressed or relapsed, and 2 patients were lost to follow-up. CONCLUSION: Ocular amyloidosis most commonly presents as an eyelid or conjunctival mass or diffuse thickening and can also present as an orbital mass. Diagnosis is mainly dependent on histopathological examination. Surgery is the main treatment and is done to confirm the diagnosis to guide further treatment, preserve function, and prevent complications that threaten visual acuity. Close postoperative follow-up is necessary.

Upper Eyelid Contour Changes After Müller's Muscle Conjunctiva Resection.

PURPOSE: To analyze the upper eyelid contour after Müller's muscle conjunctiva resection (MMCR) performed by four different surgeons. METHODS: Comparative cross-sectional analysis of the pre- and postoperative contours of a control group and four groups of upper lids (n = 88) of 65 patients who underwent MMCR at four international centers. The procedure employed was essentially the same as described by Putterman but performed with different instruments to entrap the posterior lamella. Multiple medial and lateral margin lid distances were measured on Bézier lines expressing the pre- and postoperative lid contours. RESULTS: Preoperatively, two groups had significant lateral and medial ptosis. After MMCR, the lateral segment of the lid's contour was corrected in all groups. In the two groups with more pronounced ptosis, the nasal lid contour was undercorrected. CONCLUSIONS: In MMCR, regardless of the instrument used to entrap the posterior lamella, the amount of medial tissue resection is essential to avoid postoperative nasal undercorrection. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Granzyme B Expression in Conjunctiva of Patients with Pterygium.

Pterygium is often associated with chronic ultraviolet (UV) radiation exposure and characterized by the overgrowth of conjunctiva and extracellular matrix (ECM) remodeling. Notably, several studies in the skin have demonstrated that chronic UV radiation can upregulate Granzyme B (GrB) expression and increase ECM degradation. The aim of this study was to compare GrB expression between pterygium and healthy controls and to further link this GrB expression to mast cells. Post-mortem pterygium tissues and conjunctival tissues from age-matched controls were used to assess GrB expression via immunofluorescence and microscopy. We found a significantly higher density of GrB+ cells from pterygium specimens compared to healthy controls. Furthermore, many of the GrB+ cells in pterygium specimens co-expressed tryptase, a mast cell marker. These findings suggest a role for conjunctival mast cell-secreted GrB in the pathogenesis of pterygium and highlight GrB as a possible therapeutic target in delaying or halting pterygium progression.

Congenital eyelid imbrication syndrome: A rare occurrence in Pakistan.

Congenital eyelid imbrication syndrome (CEIS) is a rare condition presenting at birth and is characterised by overriding of the upper lid on the lower lid. It is due to longer upper lid, than the lower lid. Overriding leads to spontaneous eversion of the upper lids. In our patient, examination revealed canthal tendon laxity and hyperaemia of the tarsal conjunctiva. All the rest of the structures in the eyeball and adnexa were normal. Spontaneous eversion occurred in two weeks as the upper lid grew with time. No treatment was required.

Efficacy of topical 5-Fluorouracil in the management of ocular surface squamous neoplasia: a study of 101 eyes.

OBJECTIVE: To study the efficacy and side-effect profile of topical 5-Fluorouracil (5-FU) in the treatment of ocular surface squamous neoplasia (OSSN). METHODS: Retrospective study of 101 eyes of 100 patients treated with 5-FU with one week on and 3 weeks off regimen. RESULTS: Of the 100 patients (101 eyes), the mean age at diagnosis of OSSN was 49 (median, 52 years; range, 11-87 years). History of prior intervention was noted in 6 (6%) eyes. Tumor epicenter included bulbar conjunctiva (n = 54; 53%), limbus (n = 27; 27%), and cornea (n = 20;20%). Mean number of cycles of topical 5-FU administered was 3 (median, 3; range, 1-8). Complete tumor regression was achieved with topical 5-FU in 89 (88%) eyes with a mean number of 2 cycles (median, 2; range, 1-6) of 5-FU. The remaining 12 (12%) lesions underwent additional treatment including excisional biopsy (n = 7), extended enucleation (n = 3), and topical Interferon alpha 2b (n = 2) for complete tumor control. Over a mean follow-up period of 6 months (median, 5 months; range, 1-36 months) following treatment, tumor recurrence was noted in 2 (2%) patients, and side-effects were noted in 7 (7%) eyes including conjunctival hyperemia (n = 1), punctal stenosis (n = 1), sterile keratitis (n = 4), and limbal stem cell deficiency (n = 1). CONCLUSION: Topical 5-FU is an effective non-invasive therapy for OSSN with a minimal side-effect profile.

The impact of prolonged face mask use on ocular surface health during COVID-19 pandemic: a clinical, and conjunctiva impression cytology study.

PURPOSE: To investigate the relationship between prolonged face mask use and ocular surface health utilizing conjunctival impression cytology, the Schirmer test, the tear break-up time (TBUT) test, and the ocular surface disease index (OSDI) questionnaire. METHODS: In this cross-sectional prospective study, individuals who used face masks for at least eight hours per day for at least six months were compared to healthy volunteers who used face masks for no more than one hour per day. Each participant completed an OSDI questionnaire. The Schirmer test (under anesthesia), the TBUT test, and conjunctiva impression cytology analysis according to the Nelson classification method were performed on each participant. RESULTS: This study included 102 (49 male, 53 female) face mask users with an average age of 33.29 ± 7.71 years and 110 (60 male, 50 female) healthy controls with an average age of 32.96 ± 7.10 years (p = 0.746). The total OSDI score was significantly higher in face mask users than the control group (25.18 ± 3.54 vs 9.46 ± 2.13, p < 0.001). The mean Schirmer test value and TBUT were significantly lower in the study group than the control group (p < 0.001, p < 0.001). There was a statistically significant difference between the two groups in total score and stage of the Nelson classification system (p < 0.001, and p = 0.024, respectively). All conjunctiva impression cytology assessments, including cellularity, cell-cell contact, nucleus/cytoplasma ratio, goblet cell amount, and metaplasia, revealed statistically significant deterioration in the study group compared to the control group (p < 0.001, p = 0.025, p < 0.001, p < 0.001, and p < 0.001, respectively). CONCLUSION: The prolonged use of face masks leads to dry eyes. The findings of conjunctiva impression cytology indicate the role of inflammation in the pathogenesis of mask associated dry eye.

Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

Significantly different results in the ocular surface microbiome detected by tear paper and conjunctival swab.

BACKGROUND: Great variation has been observed in the composition of the normal microbiota of the ocular surface, and therefore, in addition to differences in detection techniques, the method of collecting ocular surface specimens has a significant impact on the test results.The goal of this study is to ascertain whether the eye surface microbial communities detected by two different sampling methods are consistent and hence explore the feasibility of using tear test paper instead of conjunctival swabs to collect eye surface samples for microbial investigation. MATERIALS AND METHODS: From July 15, 2021, to July 30, 2021, nonirritating tear test strips and conjunctival swabs of both eyes were used in 158 elderly people (> 60 years old) (79 diabetic and 79 nondiabetic adults) in Xinjing Community for high-throughput sequencing of the V3-V4 region of the 16S rRNA gene. The composition of the microbial communities in tear test paper and conjunctival swab samples was analyzed. RESULTS: There was no statistically significant difference in Alpha diversity of ocular surface microorganisms represented by tear strip and conjunctival swab in diabetic group (P > 0.05), but there was statistically significant difference in Alpha diversity of ocular surface microorganisms detected by tear strip and conjunctival swab in nondiabetic group (P < 0.05). There were statistically significant differences in Beta diversity of ocular surface microorganisms detected by two sampling methods between diabetic group and nondiabetic group (P < 0.05). There were statistically significant differences in ocular surface microorganisms detected by tear strip method between diabetic group and nondiabetic group (P < 0.05), but there was no statistically significant difference in conjunctival swab method (P > 0.05). CONCLUSIONS: Tear test paper and conjunctival swabs detect different compositions of microbes through two different techniques of eye surface microbe sampling. Tear test paper cannot completely replace conjunctival swab specimens for the study of microbes related to eye surface diseases.

Alterations of conjunctival microbiota associated with orthokeratology lens wearing in myopic children.

BACKGROUND: Orthokeratology (OK) lens wear increases the risk of bacterial infection, but little is known about the microbiota of the conjunctival sac in myopic children wearing OK lenses. This study aimed to investigate the changes of conjunctival microbiota in children after treatment with OK lenses using 16 S rDNA sequencing. METHODS: Twenty-eight myopic children who had been continuously wearing OK lenses for 12 to 13 months were enrolled in this prospective study. Twenty-two gender- and age-matched myopic children who had not worn OK lenses or discontinued OK lens wear at least 1 year ago were recruited as controls. Conjunctival swabs from each participant were collected for exploration of the microbiota profiles, targeting the V3-V4 regions of the 16 S rRNA gene by MiSeq sequencing. The differences in the microbial community structure and diversity were also compared between groups. RESULTS: The bacterial alpha diversity indices in the OK lens group were not different from those in the non-wearer group (P > 0.05, Wilcoxon test), while beta diversity examined using principle coordinate analysis of unweighted UniFrac divided the two groups into different clusters. Proteobacteria, Bacteroidetes, and Firmicutes were the abundant phyla in the conjunctival sac microbiota in both groups (P < 0.05, Mann-Whitney U test). Among children in the OK lens group, the Linear discriminant analysis Effect Size identified the compositional changes in OK lens-associated bacteria. Key functional genera such as Blautia, Parasutterella, and Muribaculum were enriched, whereas Brevundimonas, Acinetobacter, Proteus, and Agathobacter decreased significantly (P < 0.05, Mann-Whitney U test). Phylogenetic investigation of communities by reconstruction of unobserved states also showed altered bacterial metabolic pathways in OK lens-associated microbiota. Moreover, using receiver operating characteristic curves, Brevundimonas, Acinetobacter, Proteus, and Agathobacter alone (the area under the curve was all > 0.7500) or in combination (the area under the curve was 0.9058) were revealed to discriminate OK lens wearers from controls. CONCLUSIONS: The relative abundance of the microbial community in the conjunctival sac of myopic children can alter after OK lens wear. Brevundimonas, Acinetobacter, Proteus, and Agathobacter may be candidate biomarkers to distinguish between OK lens wearers and non-wearers.

Assessment of hemodynamic indices of conjunctival microvascular function in patients with coronary microvascular dysfunction.

OBJECTIVE: Coronary microvascular dysfunction (CMD) is a cause of ischaemia with non-obstructive coronary arteries (INOCA). It is notoriously underdiagnosed due to the need for invasive microvascular function testing. We hypothesized that systemic microvascular dysfunction could be demonstrated non-invasively in the microcirculation of the bulbar conjunctiva in patients with CMD. METHODS: Patients undergoing coronary angiography for the investigation of chest pain or dyspnoea, with physiologically insignificant epicardial disease (fractional flow reserve ≥0.80) were recruited. All patients underwent invasive coronary microvascular function testing. We compared a cohort of patients with evidence of CMD (IMR ≥25 or CFR <2.0); to a group of controls (IMR <25 and CFR ≥2.0). Conjunctival imaging was performed using a previously validated combination of a smartphone and slit-lamp biomicroscope. This technique allows measurement of vessel diameter and other indices of microvascular function by tracking erythrocyte motion. RESULTS: A total of 111 patients were included (43 CMD and 68 controls). There were no differences in baseline demographics, co-morbidities or epicardial coronary disease severity. The mean number of vessel segments analysed per patient was 21.0 ± 12.8 (3.2 ± 3.5 arterioles and 14.8 ± 10.8 venules). In the CMD cohort, significant reductions were observed in axial/cross-sectional velocity, blood flow, wall shear rate and stress. CONCLUSION: The changes in microvascular function linked to CMD can be observed non-invasively in the bulbar conjunctiva. Conjunctival vascular imaging may have utility as a non-invasive tool to both diagnose CMD and augment conventional cardiovascular risk assessment.

Non-invasive harvesting of conjunctival cells for whole transcriptome sequencing.

The purpose of this study was to determine the feasibility of using a non-invasive technique, the EYEPRIM™ conjunctival cell impression device, to harvest sufficient RNA from conjunctival cells for the whole-transcriptome sequencing. Conjunctival cells from 40 participants were collected using an EYEPRIM™ conjunctival cell impression device. RNA was extracted from the samples, followed by library construction and transcriptome sequencing. Quality checks were performed for each technical step of the experiment, and the feasibility of this procedure was examined. RNA of sufficient yield and quality was successfully extracted following additional disruption and homogenization of the conjunctival cells and collection of two impression samples per eye. Successful library preparation and RNA sequencing were performed, with all 40 samples passing the various quality checks used for each step. In conclusion, harvesting cells from the ocular surface using an impression cytology device yields good quality and sufficient mRNA for whole transcriptome sequencing to study diseases of the eye. This technique provides a convenient alternative to using post-mortem tissues or surgical excisions.

Purinergic 2X 4 (P2X4), but not P2X7, receptors increase cytosolic [Ca2+] and stimulate mucin secretion in rat conjunctival goblet cells to maintain ocular surface health.

Ionotropic purinergic receptors (P2XRs) are activated by ATP and ATP analogs. ATP can be released through ATP-permeable channels such as the pannexin hemichannels. Upon activation, the P2XRs become permeable to Ca2+, a potent stimulator of mucin secretion in conjunctival goblet cells (CGCs). The purpose of this study was to investigate the presence and function of P2XRs in CGCs. We also examined the presence of pannexin hemichannels. Rat first passage CGCs were stained with the goblet cell marker anti-cytokeratin 7 antibody and specific antibodies to P2X1-7 receptors and pannexin 1-3. mRNA expression was determined by RT-PCR using primers specific to P2XRs and pannexins. Proteins were identified with Western blotting (WB) using the same antibodies as for immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with Fura 2-AM, exposed to agonists and antagonists, and intracellular [Ca2+] ([Ca2+]i) measured. [Ca2+]i was also measured after knock down of P2X4 and P2X7 receptor expression, and when exploiting P2XR specific characteristics. Lastly, mucin secretion was measured after the addition of several P2XR agonists. All P2XRs and pannexins were visualized with IF microscopy, and identified with RT-PCR and WB. [Ca2+]i was significantly increased when stimulated with ATP (10-7-10-4 M). Suramin, a non-selective P2XR antagonist at 10-4 M did not reduce ATP-induced peak [Ca2+]i. The potent P2X7 agonist, BzATP (10-7-10-4 M) increased the [Ca2+]i, although to a lesser extent than ATP. When measuring [Ca2+]i the effect of repeated applications of ATP at 10-5 or 10-6 M the response "desensitized" after 30-60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca2+]i increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca2+]i. ATP (10-5 M), BzATP (10-4 M) and 2MeSATP (10-5 M) induced mucin secretion. We conclude that all seven P2XRs are present in cultured rat CGCs. Of the P2XRs, only activation of the homotrimeric P2X4R appears to increase [Ca2+]i and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.

Current clinical applications of anterior segment optical coherence tomography angiography: a review.

Optical coherence tomography (OCT) is a revolutionary in vivo imaging technology that presents real-time information on ocular structures. Angiography based on OCT, known as optical coherence tomography angiography (OCTA), is a noninvasive and time-saving technique originally utilized for visualizing retinal vasculature. As devices and built-in systems have evolved, high-resolution images with depth-resolved analysis have assisted ophthalmologists in accurately localizing pathology and monitoring disease progression. With the aforementioned advantages, application of OCTA has extended from the posterior to anterior segment. This nascent adaptation showed good delineation of the vasculature in the cornea, conjunctiva, sclera, and iris. Thus, neovascularization of the avascular cornea and hyperemia or ischemic changes involving the conjunctiva, sclera, and iris has become prospective applications for AS-OCTA. Although traditional dye-based angiography is regarded as the gold standard in demonstrating vasculature in the anterior segment, AS-OCTA is expected to be a comparable but more patient-friendly alternative. In its initial stage, AS-OCTA has exhibited great potential in pathology diagnosis, therapeutic evaluation, presurgical planning, and prognosis assessments in anterior segment disorders. In this review of AS-OCTA, we aim to summarize scanning protocols, relevant parameters, and clinical applications as well as limitations and future directions. We are sanguine about its wide application in the future with the development of technology and refinement in built-in systems.

Diurnal changes and topographical distribution of ocular surface epithelial dendritic cells in humans, and repeatability of density and morphology assessment.

PURPOSE: Dendritic cells (DC) play a crucial role in ocular surface defence. DC can be visualised in vivo by confocal microscopy but have not yet been fully characterised in humans. This study investigated the diurnal variation, topographical distribution and repeatability of DC density and morphology measurements. METHODS: In vivo confocal microscopy (IVCM) was conducted on 20 healthy participants (mean age 32.7 ± 6.4 years, 50% female) at baseline and repeated after 30 minutes, 2, 6 and 24 h. Images were captured at the corneal centre, inferior whorl, corneal periphery, limbus and bulbar conjunctiva. DC were counted manually, and their morphology was assessed for cell body size, presence of dendrites, and presence of long and thick dendrites. Mixed-model analysis, non-parametric analyses, Bland and Altman plots, coefficient of repeatability (CoR) and kappa were used. RESULTS: There were no significant changes in DC density (p ≥ 0.74) or morphology (p > 0.07) at any location over the 24-h period. The highest DC density was observed at the corneal limbus followed by the peripheral cornea (p < 0.001), with the lowest density at the corneal centre, inferior whorl and bulbar conjunctiva. Most DC at the corneal periphery, limbus and bulbar conjunctiva had larger cell bodies compared with the corneal centre (p ≤ 0.01), and the presence of long dendrites was observed mostly at non-central locations. Day-to-day CoR for DC density ranged from ±28.1 cells/mm2 at the corneal centre to ±56.4 cells/mm2 at the limbus. Day-to-day agreement of DC morphology determined by kappa ranged from 0.5 to 0.95 for cell body size, 0.60 to 0.95 for presence of dendrites, and 0.55 to 0.80 for the presence of long dendrites at various locations. CONCLUSIONS: No diurnal changes are apparent in corneal or conjunctival DC. Substantial topographical differences exist in DC density and morphology. IVCM provides good repeatability of DC density and acceptable agreement of DC morphology.

Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane.

OBJECTIVES: This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies. PROCEDURES: A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression. RESULTS: Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins. CONCLUSIONS: This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.