Enhancing Ocular Surface in Dry Eye Disease Patients: A Clinical Evaluation of a Topical Formulation Containing Sesquiterpene Lactone Helenalin.

The aim of this work was to assess the tolerability, safety, and efficacy of an ophthalmic topical formulation containing helenalin from Arnica montana and hyaluronic acid 0.4% (HA) in patients with mild-to-moderate Dry Eye Disease (DED) exhibiting positive Matrix Metalloproteinase 9 (MMP-9) test results. Tolerability and safety were evaluated in 24 healthy subjects. Participants were instructed to apply one drop of the formulation three times a day in the study eye, for 2 weeks, followed by a clinical follow-up of 21 days. Efficacy was studied in 48 DED patients randomized into Study (Group 1/receiving the studied formulation) or Control (Group 2/Receiving HA 0.4% eye lubricant) groups for 1 month. Assessments included an MMP-9 positivity test, conjunctival impression cytology (CIC), Ocular Surface Disease Index (OSDI), non-invasive film tear breakup time (NIBUT), non-invasive average breakup time (NIAvg-BUT), ocular surface staining, Schirmer's test, and meibomiography. A crossover design with an additional 1-month follow-up was applied to both groups. Healthy subjects receiving the studied formulation exhibited good tolerability and no adverse events. Regarding the efficacy study, Group 1 exhibited a statistically significant reduction in the MMP-9 positivity rate compared to Group 2 (p < 0.001). Both Group 1 and Group 2 exhibited substantial improvements in OSDI and NIBUT scores (p < 0.001). However, Group 1 demonstrated a significant improvement in NI-Avg-BUT and Schirmer's test scores (p < 0.001), whereas Group 2 did not (p > 0.05). Finally, after the crossover, the proportion of MMP-9-positive subjects in Group 1 increased from 25% to 91.6%, while Group 2 showed a significant decrease from 87.5% to 20.8%. Overall, the topical formulation containing sesquiterpene helenalin from Arnica montana and hyaluronic acid was well tolerated and exhibited a favorable safety profile. Our formulation reduces DED symptomatology and modulates the ocular surface inflammatory process; this is evidenced by the enhancement of CIC, the improvement of DED-related tear film status, and the reduction of the MMP-9 positivity rate.

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.

Assessment of Mucin-Associated Gene Expression Levels on the Ocular Surface.

The ocular surface is covered with a mucus layer. The mucin-associated genes expressed in the ocular surface cells include MUC1, MUC4, MUC5AC, and MUC16. Impression cytology is useful for collecting specimens from the ocular surface, their histological examination, and measuring mucin-associated gene expression levels. The expression of mucin-associated gene levels was assessed by quantitative polymerase chain reaction. The expression levels of these mucin-associated genes are potential biomarkers for ocular surface diseases, including dry eye disease.

Evaluation of Dry Eye Severity and Ocular Surface Inflammation in Patients with Pemphigus and Pemphigoid.

PURPOSE: To evaluate ocular surface involvement, tear cytokine levels, and histopathological changes in pemphigus and pemphigoid patients. METHODS: A total of 22 patients (15 pemphigus and 7 pemphigoids) and 21 non-diseased controls were enrolled in our study. All participants underwent ocular surface evaluation, which included ocular surface disease index test, slit lamp observation, dry eye-related examination, tear multicytokine analysis, and conjunctival impression cytology. RESULTS: Pemphigus and pemphigoid patients presented much more severe conjunctivochalasis, corneal epithelial defects, corneal opacity, symblepharon   and dry eye. Severe ocular surface squamous metaplasia and a significant increase of tear macrophage inflammatory protein-1beta, tumor necrosis factor-alpha, interleukin (IL)-1ß, IL -6, and IL-8 occurred in pemphigus and pemphigoid patients. CONCLUSIONS: Our results revealed that ocular surface inflammation and dry eye persist in most pemphigus and pemphigoid patients, and do not occur in parallel with the systemic course. Regular ophthalmological examinations and local anti-inflammatory should be provided for pemphigus and pemphigoid patients.

Impression Cytologic Evaluation of the Conjunctiva in Patients Treated with Topical 1% Voriconazole

Objectives: The aim of the present study was to evaluate any conjunctival metaplastic changes by impression cytology in patients who underwent topical 1% voriconazole treatment for severe fungal keratitis. Materials and Methods: The study was conducted at Ege University Faculty of Medicine, Departments of Ophthalmology and Medical Pathology. Patients who were treated with 1% topical voriconazole for fungal keratitis for at least 3 months were included. The used topical voriconazole treatment was initiated as one drop every hour and was tapered according to clinical improvement in all patients. Treatment was continued 4 times a day for at least 3 months. Impression cytology samples were collected at least 3 months after cessation of topical voriconazole from the affected eyes and from the fellow eyes as a control group. Collected specimens were transferred to the pathology department for evaluation and grading (Nelson's grading system). Results: The mean age of the patients was 57.68±17.32 years (range, 22-87 years). The impression cytology grade of the inferior bulbar conjunctiva was 1.73±0.77 (range, 0-3) in the study group and 1.19±0.98 (range, 0-3) in the control group (p=0.03). The impression cytology grade of the temporal bulbar conjunctiva was 1.69±0.73 (range, 0-3) in the study group and 1.15±0.88 (range, 0-3) in the control group (p=0.02). The impression cytology grades of the nasal and superior bulbar conjunctiva did not differ statistically (p values 0.13 and 0.17, respectively). Conclusion: Topical voriconazole is an effective broad-spectrum antifungal drug, but it induces conjunctival squamous metaplasia. Clinicians should be aware of this possible side effect of topical voriconazole and should carefully evaluate the conjunctiva of treated patients at each visit to detect possible metaplastic changes.

Increased expression of neutrophil CD15 correlates with the severity of anterior ocular surface damage in type II diabetes mellitus.

BACKGROUND: Diabetes mellitus is associated with increased risk of ocular surface diseases in elderly. We consider neutrophil CD15 as a potential marker of ocular surface damage in type II diabetes mellitus patients. AIM: We aimed to evaluate expression of neutrophil CD15 and correlate it with results of conjunctival impression cytology and routine objective anterior ocular surface tests (TMH, NIBUT, LLT, MGD) in T2DM patients. MATERIALS AND METHODS: We prospectively enrolled sixty type II diabetes mellitus patients (120 eyes) into a study group. The control group included forty (80 eyes) age- and sex-matched healthy individuals. All patients underwent comprehensive ophthalmological examination, and tear meniscus height test (TMH), noninvasive tear break-up time (NIBUT), lipid layer thickness measurement (LLT), Meibomian gland dysfunction evaluation (MGD), conjunctival impression cytology (CIC) and expression of CD15. RESULTS: Abnormal Nelson's grades of squamous metaplasia (grades 2 and 3) were observed in 50% (60 eyes) of the study group, and 13.8 (11 eyes) of the control group. Fifteen patients with type II diabetes mellitus suffered from grade 3 squamous metaplasia. Nelson's grades of squamous metaplasia have shown a positive correlation with the level of CD15 expression either in the study and control groups (rs = 0.628, p = <0.0001; rs = 0.746, p < 0.0001; respectively). CONCLUSIONS: The research shows significantly reduced values of routine objective ocular tests in type II diabetes mellitus patients in comparison to healthy participants older than 60 y.o. Increased CD15 in the peripheral blood is associated with the development of squamous metaplasia and may be used to evaluate the severity of ocular surface damage in type II diabetes mellitus patients.

The Effect of Autografts from the Inferior and Superior Bulbar Conjunctiva on the Ocular Surface in Primary Pterygium Surgery: A Cytology Study.

Objectives: This study aimed to evaluate the effect of using an inferior or superior conjunctival autograft in primary pterygium surgery on the postoperative ocular surface. Materials and Methods: Forty eyes of 40 patients who underwent pterygium surgery with autograft were included in the study. Cytological cell counts were performed on samples taken from the bulbar conjunctiva by impression cytology before and 1 year after the operation. Schirmer 1 test score, lissamine green conjunctival staining score, tear film break-up time (TBUT), and fluorescein corneal staining scores were evaluated. The pain levels of the patients were evaluated with visual analog scale at postoperative 1 day and 1 week. Results: Corneal and conjunctival staining, TBUT, and Schirmer test results demonstrated significant improvement in all patient groups after surgery, but there was no difference between groups (p>0.05). In both preoperative and postoperative impression cytology, the number of goblet cells in the inferior bulbar conjunctiva was higher than in the superior bulbar conjunctiva (p<0.001), while there was no such difference in epithelial cell or mucin staining. There were no significant cytological changes postoperatively in either group (p>0.05). Conclusion: Pterygium surgery with autografting improved tear function tests regardless of graft location. Goblet cell count was higher in the inferior bulbar conjunctiva than in the superior bulbar conjunctiva in both postoperative and preoperative impression cytology. However, there was no significant difference in postoperative epithelial and goblet cell counts or mucin staining between the groups before and after surgery. We think that using the inferior bulbar conjunctiva is an appropriate choice in cases where the superior conjunctiva cannot be used as a graft or when future glaucoma surgery is possible.

Conjunctival Impression Cytology and Tear Film Changes in Sarcoidosis: A Subjective and Objective Diagnosis Study.

Objectives: To evaluate sarcoidosis-induced tear film changes using subjective and objective diagnostic tests, particularly conjunctival impression cytology (IC), and to compare the results with healthy individuals. Materials and Methods: This study evaluated clinical data collected between January 2019 and January 2021 from 57 right eyes of 57 sarcoidosis patients without ocular involvement (Group 1) and 33 right eyes of 33 healthy individuals with similar demographic characteristics (Group 2). The Schirmer I test, tear break-up time (TBUT), fluorescein staining, and conjunctival IC were all performed as part of the conjunctival and corneal examinations following a thorough ophthalmological examination. The Ocular Surface Disease Index (OSDI) was used to assess subjective ocular symptoms. Results: The mean ages in Groups 1 and 2 were 49.26±3.18 and 51.91±2.89 years, respectively (p=0.720). The mean Schirmer I test, TBUT, and OSDI scores differed significantly (p<0.05 for all), with Group 1 having a significantly higher percentage of dry eyes than Group 2. Group 1 had significantly higher Nelson's grading system grades than Group 2 based on conjunctival IC analysis (p=0.001). There were no significant differences in visual acuity (p=0.17) or intraocular pressure (p=0.14) between groups. Conclusion: Sarcoidosis patients had significantly higher Nelson grades in conjunctival IC, as well as significantly higher percentages of dry eye as determined by the Schirmer I test, TBUT, and OSDI. Reduced tear quantity and quality may destabilize the tear film layer, resulting in a variety of ocular symptoms.

Ocular surface involvement and histopathologic changes in the acute stage of Stevens-Johnson syndrome and toxic epidermal necrolysis: a cross-sectional study.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare and extremely serious drug-induced dermatological disorders. The ocular surface condition at the early stage has been little studied and should contribute to novel perspectives in early and effective topical therapy of these diseases. The objectives of the study were to evaluate the acute phase of ocular surface involvement and histopathologic changes in patients with acute SJS/TEN. METHODS: Ten patients with acute phase of SJS/TEN onset and eleven age- and sex-matched healthy volunteers were recruited. Ocular surface symptoms and signs, conjunctival impression cytology, and tear multi-cytokine were assessed. RESULTS: Ocular surface objective signs were normal at the acute stage of SJS/TEN, while most patients have abnormal ocular surface subjective symptoms and meibomian gland secretion. Conjunctival impression cytology showed a significant decrease in goblet cell density and severe ocular surface squamous metaplasia in acute SJS/TEN patients. Tear multi-cytokine analysis showed all 21 pro- and anti-inflammatory cytokines all sharply elevated. Goblet cell density was significantly negatively correlated with tear C-X3-C motif chemokine ligand 1 (CX3CL1) and interleukin 13. CONCLUSIONS: Severe pathologic squamous metaplasia and inflammation onset in the ocular surface at the acute stage of the SJS/TEN, even if the ocular surface condition seemed basically normal with adequate systemic immunosuppressant and general supportive treatment. Early topical anti-inflammatory therapy should be carried out actively.

[Evolution of dry eye disease diagnostics]. / Evolyutsiya metodov diagnostiki sindroma sukhogo glaza.

This article reviews modern functional and instrumental examination methods included in the diagnostic algorithm for dry eye disease. The described methods can serve as an objective criterion for the effectiveness of the therapy.

Prevalence of dry eye, its categorization (Dry Eye Workshop II), and pathological correlation: A tertiary care study.

Purpose: To study the prevalence of dry eye disease (DED), further categorize using DEWS II protocol, grade squamous metaplasia in each group, and determine associated risk factors in a tertiary care hospital. Methods: This cross-sectional hospital-based study screened 897 patients ≥30 years via systematic random sampling. Patients with both symptoms and signs as defined by the Dry Eye Workshop II protocol were considered as DED, further categorized, and subjected to impression cytology. Categorical data were assessed using the Chi-square test. P value < 0.05 was considered statistically significant. Results: In total, 265 (of 897) patients were defined as DED based on the presence of symptoms (DEQ-5 ≥6) and at least one positive sign (fluorescein breakup time [FBUT] <10 s or OSS ≥4). DED prevalence was thus 29.5% with aqueous deficient dry eye (ADDE), evaporative dry eye (EDE), and mixed type seen in 92 (34.71%), 105 (39.62%), and 68 (25.7%) patients, respectively. The risk of developing dry eye was higher in the age above 60 years (33.74%) and in the third decade. Females, urban dwellers, diabetics, smokers, history of previous cataract surgery, and usage of visual display terminal devices were found to be significantly associated with risk factors of DED. Squamous metaplasia and goblet cell loss were more severe in mixed compared to EDE and ADDE. Conclusion: Hospital-based prevalence of DED is 29.5% with a preponderance of EDE (EDE 39.62%, ADDE 34.71%, and mixed 25.71%). A higher grade of squamous metaplasia was seen in the mixed type compared to other sub-types.

Comparing the efficacy of sodium hyaluronate eye drops and carboxymethylcellulose eye drops in treating mild to moderate dry eye disease.

Purpose: Aim of this study was to compare the efficacy of sodium hyaluronate (SH) and carboxymethyl cellulose (CMC) eye drops in treating mild to moderate dry eye disease in terms of relief of symptoms, mean change in tear film breakup time, Schirmer's test, and impression cytology of conjunctiva from baseline. Methods: An observational study was carried out during a 2-year period in our tertiary referral hospital. The study consisted of 60 patients randomly allotted to two groups to receive SH and CMC eye drops for an 8-week period. Ocular surface disease index, tear film breakup time, and Schirmer's test were performed at baseline visit and 4 and 8 weeks of treatment, and impression cytology of conjunctiva was performed at baseline and 8 weeks. Results: Significant improvement in patient symptoms, tear film breakup time, and Schirmer's test from baseline was seen in both SH and CMC groups at 8 weeks posttreatment, whereas impression cytology of conjunctiva in both groups did not show significant improvement at 8 weeks of treatment. Data analysis using unpaired t-test showed comparable results. Conclusion: Both CMC and SH demonstrated equal efficacy in treating mild to moderate dry eye disease.

Comparison of Adhesive Tape Impression Cytology, Hair Plucks, and Fungal Culture for the Diagnosis of Dermatophytosis in Dogs and Cats.

Rapid diagnosis of dermatophytosis is essential for early treatment induction and prevention of spreading to other animals and humans. No single diagnostic test is identified as the "gold standard". The purpose of the study was to evaluate the sensitivity of adhesive tape impression (ATI) cytology in dermatophyte identification and to compare three diagnostic tests for dermatophytosis. Thirty dogs, with alopecia (n = 19) or kerion (n = 11), and fifteen cats with alopecia were included in the study. Dermatophytosis was diagnosed with tape preparations in 82.2% (37/45) of cases, while with hair plucks in 66.7% (30/45) and fungal culture in 80% (36/45). In kerions, tape preparations and fungal culture had the same sensitivity (10/11, 90.9%) that was higher than that of hair plucks (4/11, 36.4%). The sensitivity was higher in cats than in dogs with alopecia for all tests, 80% versus (vs.) 73.7%, 86.7% vs. 68.4%, and 93.3% vs. 68.4% for cats and dogs for hair plucks, fungal culture, and tape preparations, respectively. No significant difference was found between the three tests, except for dogs with kerion. Hair plucks were less sensitive than fungal culture in kerions (p = 0.041), while in comparison with tape preparations they were marginally not significantly different (p = 0.078). ATI cytology is a useful diagnostic test in dermatophytosis, in dogs with kerion as well as cats.

Diagnostic Algorithm for Surgical Management of Limbal Stem Cell Deficiency.

BACKGROUND: Limbal stem cell deficiency (LCSD) presents several challenges. Currently, there is no clearly defined systematic approach to LSCD diagnosis that may guide surgical tactics. METHODS: The medical records of 34 patients with LSCD were analyzed. Diagnostic modalities included standard (visometry, tonometry, visual field testing, slit-lamp biomicroscopy with corneal fluorescein staining, Schirmer test 1, ultrasonography) and advanced ophthalmic examination methods such as anterior segment optical coherence tomography, in vivo confocal microscopy, impression cytology, and enzyme-linked immunoassay. RESULTS: Standard ophthalmological examination was sufficient to establish the diagnosis of LSCD in 20 (58.8%) cases, whereas advanced evaluation was needed in 14 (41.2%) cases. Depending on the results, patients with unilateral LSCD were scheduled to undergo glueless simple limbal epithelial transplantation (G-SLET) or simultaneous G-SLET and lamellar keratoplasty. Patients with bilateral LSCD with normal or increased corneal thickness were enrolled in the paralimbal oral mucosa epithelium transplantation (pLOMET) clinical trial. CONCLUSIONS: Based on the diagnostic and surgical data analyzed, the key points in LSCD diagnosis were identified, helping to guide the surgeon in selecting the appropriate surgical procedure. Finally, we proposed a novel step-by-step diagnostic algorithm and original surgical guidelines for the treatment of patients with LSCD.

Evaluation and comparison of subjective and objective anterior ocular surface damage in patients with type 2 diabetes mellitus and dry eye disease.

PURPOSE: To evaluate anterior ocular surface damage in patients with type 2 diabetes mellitus and dry eye disease in comparison to non-diabetic controls based on conjunctival impression cytology, objective scales (Efron, Oxford) and OSDI, to correlate vision-related quality of life with grades of squamous metaplasia in T2DM patients suffering from DED. METHODS: All participants underwent complete ophthalmologic examination including Shirmer test, TBUT, conjunctival/corneal staining (Oxford scheme), evaluation of conjunctival redness (Efron grading scale), and conjunctival impression cytology (Nelson's scale). The OSDI questionnaire was completed by both groups of patients to assess severity of DED and QoL. RESULTS: Squamous metaplasia was observed in 94% of the study group and 19.3% of controls (p = 0.0000). Based on the OSDI scores, 73.5% of patients reported mild DED and 26.5% suffered from moderate DED in the study group. The mean OSDI score for the study group with Nelson's grade 2 was 18 ± 3.52 and 20.8 ± 4.68 for Nelson's grade 3, respectively (p = 0.0745). Hence, no significant difference in QoL between grade 2 and grade 3 of squamous metaplasia was observed in patients of the study group. CONCLUSION: Impression cytology is a reliable minimally invasive tool for an accurate evaluation of the ocular surface damage in patients with DED and type 2 diabetes mellitus. Severe squamous metaplasia (Nelson's grade 3) was observed in 29.4% (10/34) of T2DM patients. In contrast, it was not detected in the control group (p = 0.0032). The absence of goblet cells in T2DM patients nether significantly reduces QoL nor contributes to the subjective DED severity (OSDI) due to complex pathways leading to DED. Thus, diagnosis of DED severity should not be solely based on subjective symptoms in this population.

Activation of Conjunctiva-Associated Lymphoid Tissue in Diabetic Patients.

PURPOSE: To evaluate changes in conjunctiva-associated lymphoid tissues (CALTs) in patients with type 2 diabetic mellitus (T2DM). METHODS: Thirty-two patients with T2DM and 32 healthy volunteers underwent comprehensive examinations. In vivo confocal microscopy and Image J were used to observe and evaluate the patients' CALT-related parameters. Conjunctival impression cytology (CIC) samples of the tarsal conjunctiva were collected from the patients, and CD4+ and CD8+ cells were evaluated by immunofluorescence staining. RESULTS: The diabetes group showed higher diffuse lymphocyte density(p < .001), follicular density(p < .001) and parafollicular lymphocyte density(p < .001). The percentages of CD4+ cells (p < .001) and CD8+ cells (p < .001) in the diabetes group were higher than those in the control group. CALT-related parameters of the diabetic patients with diabetic retinopathy showed higher degrees of activation than those of the diabetic patients without diabetic retinopathy. CONCLUSIONS: CALT activation is observed in patients with T2DM, and the activation is more obvious in patients with diabetic retinopathy. TRIAL REGISTRATION NUMBER: Retrospectively registered, ChiCTR2100046030.

Comparison of Transcriptomic Analysis of the Conjunctiva in Glaucoma-Treated Eyes with Dry Eyes and Healthy Controls.

Ocular surface disease (OSD) associated with topical glaucoma drugs is a common issue impacting treatment adherence. We aimed to identify conjunctival transcriptomic changes in glaucoma and dry eye patients, comparing them to healthy controls. Bulbar conjunctival specimens were collected via impression cytology from 33 patients treated for glaucoma, 9 patients with dry eye, and 14 healthy controls. RNA extraction and bulk RNA sequencing were performed, followed by bioinformatics analysis to detect gene dysregulation. Ingenuity pathways analysis (IPA) identified pathways and biological processes associated with these transcriptomic changes. Sequencing analysis revealed 200 modified genes in glaucoma patients compared to healthy individuals, 233 differentially expressed genes in dry eye patients versus controls, and 650 genes in treated versus dry eye samples. In glaucoma patients, 79% of altered pathways were related to host defense, while dry eye patients showed a 39% involvement of host response, 15% in cellular proliferation and integrity, and 16% of mitochondrial dysfunction. These findings were validated through qRT-PCR. Glaucoma patients showed an intensified conjunctival immune response as a potential cause of OSD, whereas in dry eye patients, in addition to the immune response, other mechanisms such as mitochondrial dysfunction or reduced cellular proliferation were observed.

[Visualization of normal ocular surface microflora via impression cytology sample using scanning electron microscopy with lanthanide contrasting]. / Vizualizatsiya normal'noi mikroflory glaznoi poverkhnosti posredstvom impressionnoi proby s ispol'zovaniem skaniruyushchego elektronnogo mikroskopa i lantanoidnogo kontrastirovaniya.

PURPOSE: To determine the possibilities of impression cytology (IC) with subsequent visualization of the sample on a scanning electron microscope in assessment of normal microflora of the ocular surface. MATERIALS AND METHODS: The article presents a visual characteristic of the microorganisms of the ocular surface (OS) captured during impression cytology (IC) in individuals without signs of inflammatory and degenerative eye diseases. The original method of staining the sample with heavy metal salts made it possible to identify the individual signs of the microorganisms in their subsequent visualization by scanning electron microscopy (SEM). RESULTS: The paper presents photomicrographs of the microorganisms most common for the OS obtained with the help of SEM, confirming and supplementing the data of non-visual methods of studying the ocular microflora. It was shown that the detection frequency of the microbial component of the OS by the visual method presented in this study is comparable with the detection frequency when using the microbial cultivation method (<80%). Coccoid and rod-shaped microorganisms were detected with relatively equal frequency, with the coccoid organisms mainly represented in association with epithelial cells. The morphological diversity of rod-shaped microorganisms is shown. CONCLUSION: The results of the study can be used as a visual reference for the normal microbiome of the eye.

Quantitation of Pax-6 protein in ocular impression cytology samples using an electrochemiluminescence immunoassay.

Paired box protein Pax-6 (oculothrombin) is a transcription factor that plays an important regulatory role in ocular, brain, and pancreatic development. Mutations of the PAX6 gene cause aniridia and Peters anomaly. Reduction in Pax-6 protein is also associated with ocular diseases such as dry eye. An electrochemiluminescence immunoassay method using the Meso Scale Discovery platform was developed to measure Pax-6 protein levels in corneal epithelial cells obtained by impression cytology. Impression cytology involves harvesting ocular epithelial cells by applying a polyethersulfone membrane patch briefly to the ocular surface using a commercially available EYEPRIM™ device. The epithelial cells that adhere to the membrane patch of the EYEPRIM™ device provide a biological sample which can be assayed for Pax-6 protein levels. Assay development identified an antibody pair capable of detecting purified recombinant Pax-6 protein produced in mammalian cells. The optimized assay has a dynamic range of 24 pg mL-1 to 100,000 pg mL-1 and a lower limit of quantification of 24 pg mL-1. Assay selectivity was demonstrated using either HeLa or HEK293 cells transfected with inhibitory RNA. Finally, the method was validated by measuring Pax-6 protein levels in impression cytology acquired samples obtained using the EYEPRIM™ device from rabbit cornea.