Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-Responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment.

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30-50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

A Comparative Genome-Wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells.

Aim: To investigate genes and pathways involved in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. Methods: Using paired human donor eyes, human organ-cultured anterior segment (HOCAS) was established in one eye to characterize GC responsiveness based on intra ocular pressure (IOP) change and, in the other eye, primary HTM cell culture was established. For RNA sequencing, total RNA extracted from GC-responder (GC-R) and non-responder (GC-NR) cells after dexamethasone (DEX) or ethanol (ETH) treatment for 7d was used. Differentially expressed genes (DEGs) were compared among five groups and validated. Results: In total, 616 and 216 genes were identified as significantly dysregulated in Group #1 and #2 (#1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR), respectively. Around 80 genes were commonly dysregulated in Group #3 (overlapping DEGs between #1 and #2), whereas 536 and 136 genes were uniquely expressed in GC-R (#4) and GC-NR HTM (#5) cells, respectively. Pathway analysis revealed that WNT signaling, drug metabolism cytochrome p450, cell adhesion, TGF-ß signaling, and MAPK signaling were associated with GC responsiveness. Conclusion: This is the first study reporting distinct gene signatures and their associated pathways for GC-R and GC-NR HTM cells. WNT and MAPK signaling are potential therapeutic targets for the management of GC-induced glaucoma.

Short and long-term effect of dexamethasone on the transcriptome profile of primary human trabecular meshwork cells in vitro.

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma.

The utility of relative afferent pupillary defect as a screening tool for glaucoma: prospective examination of a large population-based study in a south Indian population.

BACKGROUND: Previous authors have suggested that an afferent pupillary defect (APD) may serve as an effective screening tool for some specific eye diseases, especially glaucomatous optic neuropathy, since the disease usually presents asymmetrically. Its success as a screening tool for glaucoma has never been previously prospectively evaluated in a large population. METHODS: In this study, the authors carry out assessments for the presence of APD as it relates to the diagnosis of glaucoma in an existing population-based eye study in southern India. The authors calculate the sensitivity and specificity for APD, both at the level of a village/household screening and in a more comprehensive/hospital setting, as it pertains to its ability to predict glaucomatous optic neuropathy. RESULTS AND DISCUSSION: The authors find that APD assessed via the swinging flashlight test is a poor screening tool for glaucoma in this setting.

Population-based prevalence of uveitis in Southern India.

PURPOSE: To estimate the prevalence of uveitis in rural Tamil Nadu, India. METHODS: Cluster sampling identified a population-based sample of 5150 persons ages 40 years and older, representative of three districts in Tamil Nadu, India. Each received a comprehensive dilated ocular examination by an ophthalmologist, and results were registered on forms requiring responses about the presence of ocular inflammatory signs and inflammatory diagnoses. Potential uveitis cases were confirmed by consensus of two uveitis specialists, upon review of study and medical records. Crude prevalence rates and prevalence rates age-adjusted to reflect the 2001 Tamil Nadu census population were calculated. RESULTS: Crude and age-adjusted prevalence rates for endogenous uveitis were 310/100,000 and 317/100,000. The corresponding rates for all ocular inflammation were 450 and 467/100,000. Males tended to have a greater prevalence than females in this population, and older persons tended to have higher prevalence than younger persons. The majority of cases of posterior uveitis and infectious endophthalmitis were visually compromised, but few among the cases of other forms of ocular inflammation were visually impaired. CONCLUSIONS: The results suggest that nearly one in 200 persons in rural, South India has been affected by ocular inflammation in at least one eye by mid to late adulthood, about one in 330 if cases related to surgery or trauma are not included. Postsurgical endophthalmitis and posterior uveitis were associated with a high rate of vision loss. These results indicate that uveitis is an important cause of ocular morbidity and of vision loss in this population.

Relationship between vision impairment and eye disease to vision-specific quality of life and function in rural India: the Aravind Comprehensive Eye Survey.

PURPOSE: To determine the impact of vision impairment and eye diseases on vision-specific quality of life and visual function in an older population of rural southern India. METHODS: Presenting and best-corrected visual acuity and burden of eye diseases were determined in a population aged 40 years and older, identified through a random cluster sampling strategy from 50 villages of rural south India. A questionnaire validated previously for use in this population was used to ascertain quality of life and visual function. Visual acuity measurements were obtained with illiterate E Early Treatment Diabetic Retinopathy Study (ETDRS) charts. Cataract was graded and defined based on the Lens Opacities Classification System (LOCS) III. Macular degeneration was defined based on the classification system proposed by the International ARM Epidemiologic Study Group. Glaucoma was defined based on results of clinical examinations including optic disc and visual fields. Analyses were performed to explore the relationship of overall and subscale quality-of-life and visual function scores with presenting acuity in the better-seeing eye, specific eye diseases, and demographic variables. RESULTS: Information on quality of life and visual function were available for 5119 (99.4%) of 5150 study subjects. The mean presenting visual acuity in the better eye was 0.76 +/- 0.53 logMAR (logarithm of the minimum angle of resolution) units. Age, education, occupation, presenting acuity in the better eye, and presence of a cataract, glaucoma, or refractive error were independently associated with overall quality-of-life and vision function scores. After adjustment for demographic variables and ocular disease, persons with vision impairment or bilateral blindness based on presenting visual acuity had lower scores across all domains of quality of life and vision function. Scores for subscales of quality-of-life and vision function domains were significantly lower among those with age-related cataract and glaucoma compared with persons without those eye diseases. CONCLUSIONS: Presenting vision in the better eye was associated with quality of life and vision function in this older population of rural south India. Subjects with glaucoma and age-related cataract had an associated decrease in quality of life and vision function, independent of presenting visual acuity in the better eye.

Female reproductive factors and eye disease in a rural South Indian population: the Aravind Comprehensive Eye Survey.

PURPOSE: To determine the potential associations of female reproductive factors with age-related cataract, open-angle glaucoma, macular degeneration, and myopia in an older population of rural south India. METHODS: This was a population-based, cross-sectional study of older adults in rural south India identified through a cluster sampling technique. Histories relating to female reproductive factors were ascertained through a questionnaire administered by trained workers. Detailed ocular examinations including automated perimetry were performed on all participants at a base hospital to arrive at a diagnosis of ocular morbidity. RESULTS: The study achieved a high response rate (93.0%), with examinations performed on 5150 of the eligible 5539 persons aged 40 years or more. Age at menarche was available for 2797 (98.6%) of the women and age at natural menopause for 1841 (98.0%) of 1878 women who were postmenopausal. The mean age at menarche was 14.8 +/- 1.8 years, and the mean age at menopause was 43.4 +/- 3.9 years. The mean duration of endogenous estrogen exposure was 28.4 +/- 4.3 years. The median number of pregnancies was 4 (mean, 4.3 +/- 2.6; range, 0-16). Older age at menarche (>or=14 years) was associated with reduced risk for age-related cataract and myopia, and greater risk for macular degeneration. Neither age at menopause nor duration of endogenous estrogen exposure was associated with any of the ocular diseases studied. Parity was not associated with any of the ocular diseases studied in a multivariate model. CONCLUSIONS: Female reproductive factors do not appear to influence age-related cataract, open-angle glaucoma, macular degeneration or myopia significantly in rural south India.

Ocular trauma in a rural south Indian population: the Aravind Comprehensive Eye Survey.

PURPOSE: To determine the rate of ocular trauma in a rural population of southern India and its impact on vision impairment and blindness. METHODS: A population-based cross-sectional study of 5150 persons 40 years or older in a randomly chosen rural population of 3 districts of southern India. Prospective information on trauma, type and agent of injury, setting of injury, and details of treatment sought for the last episode was recorded with questionnaires after face-to-face interviews. All interviewed subjects underwent a comprehensive ocular examination, including vision estimations, slit-lamp biomicroscopy examinations, and dilated posterior segment examinations. RESULTS: We elicited a history of ocular trauma in either eye from 229 (4.5%) persons, including 21 (0.4%) persons with bilateral ocular trauma. Blunt injuries (n = 124; 54.9%) were the major cause for trauma reported in this population. The most common setting where the ocular trauma occurred was during agricultural labor (n = 107; 46.9%). Nearly three quarters (n = 170; 74.2%) of those reporting ocular trauma sought treatment from an eye specialist (n = 104; 57.8%) and one fifth (n = 37; 20.6%) from a traditional healer. The age-adjusted (adjusted to the population estimates for India for the year 2000) prevalence for blindness in any eye caused by trauma was 0.8% (95% confidence interval [CI], 0.4-1.1). The odds ratios (OR) for trauma were higher for males (OR, 2.2; 95% CI, 1.6-3.0) and laborers (OR, 1.7; 95% CI, 1.2-2.4) and lower for literates (OR, 0.7; 95% CI, 0.5- 0.9). Seeking treatment from a traditional eye healer for trauma was not associated with vision impairment (OR, 1.0; 95% CI, 0.3-3.2) or with blindness (OR, 3.4; 95% CI, 0.2-56.5). CONCLUSIONS: Eye care programs may need to consider ocular trauma as a priority in this population, because the lifetime prevalence of ocular trauma is higher than that reported for glaucoma, age-related macular degeneration, or diabetic retinopathy from this population. Simple measures such as education regarding the use of protective eyewear could possibly significantly decrease this preventable cause of visual disability.

Prevalence of vitreoretinal disorders in a rural population of southern India: the Aravind Comprehensive Eye Study.

OBJECTIVE: To determine the magnitude of vitreoretinal disorders in a rural southern Indian population. METHODS: Cluster sampling was used to identify individuals 40 years and older in Tamil Nadu in southern India. Demographic details, vision measurement and refraction using logMAR charts, anterior segment slitlamp examination, dilated posterior segment slitlamp examination using a 78-diopter (D) lens, and indirect ophthalmoscopy using a 20-D lens were performed. RESULTS: Complete retinal data were available for 4917 (95.5%) of the 5150 persons examined. The prevalence of any vitreoretinal disorder was 10.4% (95% confidence interval [CI], 9.5%-11.3%). The population prevalence of bilateral blindness among persons with vitreoretinal disorders was 0.3% (95% CI, 0.2%-0.5%). The prevalence of diabetic retinopathy was 0.5% (95% CI, 0.3%-0.7%) in the general population and 10.5% (95% CI, 6.5%-14.5%) in patients with diabetes mellitus. Only 6.7% of individuals with diabetic retinopathy had previous ophthalmic examinations. The prevalences of early and late age-related macular degeneration were 2.7% (95% CI, 2.2%-3.2%) and 0.6% (95% CI, 0.4%-0.8%), respectively. CONCLUSIONS: Vitreoretinal diseases appear to be a major public health problem in India. Emphasis on diabetic screening, diabetic therapy, and appropriate laser therapy of diabetic retinopathy must be explored.

The utilization of eye care services by persons with glaucoma in rural south India.

PURPOSE: To determine utilization of eye care services, in particular those relating to glaucoma, in a rural population of southern India aged 40 years or older. METHODS: A total of 5,150 subjects aged 40 years or older selected through a random cluster sampling technique from three districts in southern India underwent detailed ocular examinations for vision impairment, blindness, and ocular morbidity. Information regarding previous use of eye care services was collected from this population through a questionnaire administered by trained social workers prior to ocular examinations. RESULTS: One thousand eight hundred and twenty-seven persons (35.5%) gave a history of prior eye examinations, primarily from a general hospital (n = 1,073, 58.7%). Increasing age and education were associated with increased utilization of eye care services. Among the 3,323 persons who had never sought eye care, 912 (27.4%) had felt the need to have an eye examination but did not do so. Only one third of persons with vision impairment, cataracts, refractive errors, and glaucoma had previously utilized services. Of the 64 subjects diagnosed as having primary open-angle glaucoma, 32 (50%) had previously seen an ophthalmologist, but none had had an eye examination within 1 year before the study. Only six (19%) of the 32 had been diagnosed as having glaucoma (9% of all subjects found to have glaucoma in the survey). Thirteen (20.3%) of the 64 subjects were blind in either eye due to glaucoma, including one person who was bilaterally blind. CONCLUSIONS: A large proportion of persons in a rural population of southern India who require eye care are currently not utilizing existing eye care services. Strategies to improve the uptake of services are required to reduce the burden of blindness due to glaucoma in southern India.

Lens opacities in a rural population of southern India: the Aravind Comprehensive Eye Study.

OBJECTIVES: To determine the prevalence of lens opacities in an older population in rural southern India. METHODS: A cross-sectional study of 5150 persons aged 40 years and more who were selected as part of a representative sample from three southern districts of the state of Tamil Nadu in southern India. All lenses were graded and classified for opacities and other disorders by slit lamp after pupillary dilation, using the Lens Opacification Classification System (LOCS) III and standard photographs. Definite cataract was defined as either LOCS III nuclear opalescence of grade 3.0 or more and/or cortical cataract of grade 3.0 or more and/or posterior subcapsular cataract (PSC) of grade 2.0 or more. RESULTS: Definite cataract was present in one or both eyes in 2499 (47.5%) of 5150 subjects. The age-adjusted prevalence (adjusted to U.S. population estimates for 2000) of definite cataract in this population was 61.9% (95% CI, 60.6-6.3). The prevalence was significantly lower in men (P=0.0002). In those eyes with cataracts, nuclear cataract (59.7%) was most common, and cortical cataract was present in 20.0% and PSC in 24.3%. Cataract surgery had been performed in 9.4% persons, including 3.0% who had bilateral surgery for cataracts. Best corrected visual acuity was less than 6/60 in 3.7% of aphakic eyes and in 0.8% of pseudophakic eyes. CONCLUSIONS: The higher age-adjusted prevalence and relatively early onset of age-related cataract in this population suggest that the cataract-centered approach to minimizing preventable blindness, adopted by the National Program for Prevention of Blindness in India, is appropriate.

Enzymatic sclerostomy: pilot human study.

OBJECTIVE: To evaluate the feasibility and safety of enzymatic sclerostomy as a new modality to lower intraocular pressure in patients with open-angle glaucoma. METHODS: This single-center, prospective, noncomparative, interventional case series included 15 blind symptomatic eyes of 15 patients with primary open-angle glaucoma. Enzymatic sclerostomy was performed with the patient under topical or peribulbar anesthesia. A specially designed polymethylmethacrylate enzyme applicator filled with a mean +/- SD of 123 +/- 13 microg of collagenase was introduced through a 5-mm peritomy, and affixed to the limbus by means of cyanoacrylate tissue glue. After 22 to 24 hours, the applicators were removed and the patients were followed up for 1 year. Intraocular pressure changes from baseline and complications related to the procedure were the main outcome measures. RESULTS: Controlled thinning of the treated sclera associated with aqueous percolation and shallow filtration bleb was seen in all eyes in the immediate postoperative period. The mean +/- SD intraocular pressure decreased from 43.5 +/- 9.8 mm Hg (while the patients were receiving a mean +/- SD of 1.75 +/- 0.75 antiglaucoma medications) preoperatively to 24.8 +/- 10.6 mm Hg (a 43.0% decrease from baseline with no antiglaucoma medication) on the first postoperative day and to 34.8 +/- 10.5 mm Hg (a 20.0% decrease from baseline with no antiglaucoma medication) at the end of 1 year. Ophthalmic adverse effects were limited to the treated area and included immediate postoperative transient conjunctival reaction ranging from mild chemosis to conjunctival maceration. Immediate full-thickness perforation developed in 1 eye; the patient was treated and excluded from data analysis. Two eyes developed symptoms related to increase in intraocular pressure after 9 months; the patients were treated and excluded from further data analysis. No systemic complications were noted. CONCLUSIONS: Enzymatic sclerostomy demonstrated immediate and sustained intraocular pressure reduction and provided symptomatic relief in blind eyes with primary open-angle glaucoma. The procedure, however, needs further technical refinement.