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Background Loss of cells in the human trabecular meshwork (TM) has been reported with ageing and in glaucoma. This study aims to identify, quantify and determine the age-related changes of human TM stem cells (TMSCs). Methods Isolation of TM cells/ paraffin sectioning was carried out using human corneoscleral rings and whole globes. The TM cells/ sections were immunostained for the stem cell markers ATP-binding cassette protein G2 (ABCG2), nerve growth factor receptor p75 and AnkyrinG (AnkG). Images were acquired using Leica SP8 confocal microscope. The isolated cells were analyzed for two parameters- ABCG2 expression and nucleus to cytoplasmic ratio (N/C ratio). The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified. Spearman rank order correlation was used to determine the association between age and the cell counts. Results The TMSCs were identified based on two parameters- high ABCG2 expression and high N/C ratio > 0.7. These stem cells were also positive for p75 and AnkG. The TMSC content based on the two parameters was 21.0 ± 1.4% in < 30 years age group, 12.6 ± 6.6% in 30–60 years and 4.0 ± 3.5% in > 60 years. The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe’s line region of the TM. A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing. Conclusion The human TMSCs were identified and quantified based on two parameter analysis. This study established a significant association between age-related reduction in TMSC content and TM cell loss.

Purpose:To compare the structural integrity and functional status of the donor corneas stored in Cornisol and Optisol-GS.Methods:Fifteen optical grade corneal donor buttons (6 pairs; 3 individual) obtained from Rotary Aravind International Eye Bank were used for the study. The left eye of the paired sample was preserved in Cornisol and the right in Optisol-GS. The three individual buttons were used for the baseline data. The corneas were assessed with slit lamp and specular microscope before and after storage time (7, 10, or 14 days). They were then immunostained for markers of structural integrity (ZO-1, Phalloidin) and functionality (Na+/K+ ATPase). The images were acquired using confocal microscope and analyzed using ImageJ software.Results:There was no difference in the clinical evaluation of the corneal layers between the two media. No marked variation was observed in the immunostaining data with reference to the storage period. Intact cellular integrity was identified in 91% (51%, 98%) [Median (min, max)] of cells in Cornisol and 94% (38%, 98%) cells in Optisol based on ZO-1 staining, comparable to the baseline data [87% (76%, 97%)]. Stress fibers were detected in 42.5% (1%, 88%) cells in Cornisol stored corneas and in 55% (11%, 94%) in Optisol when stained for actin cytoskeleton, which correlated with the presence of epithelial defect before storage and vacuolated endothelial cells after storage. No difference was observed between the two media based on the staining pattern for Na+/K+ ATPase.Conclusion:Cornisol and Optisol-GS are equivalent in maintaining the structural integrity and functionality of the donor corneas.

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