Detection of Cellular Senescence in Conjunctival Epithelium from Elderly Individuals and Role of Ocular Senescent Cells in the Disruption of Barrier Function

Abstract

Purpose : Although changes in the ocular surface microenvironment are known to occur during aging, it remains unclear whether cellular senescence at the ocular surface contributes to age-related phenotypes. Here, conjunctival tissue samples obtained from elderly subjects (aged 65 years or older) and irradiated primary conjunctival epithelial cells (CjECs) were used to determine the potential role of ocular senescent cells.

Methods : Our study involved 10 conjunctival tissue samples obtained from 10 healthy elderly subjects [4 males and 6 females; mean age: 68.5 years (range: 65-81 years)], and primary CjECs obtained from human donor eyes that were cultured and X-irradiated at 10 Gy. Tissue samples and irradiated CjECs were examined using immunostaining or western blotting for p16^INK4a expression (an important marker of cellular senescence) and for the senescence-associated beta-galactosidase (SA-βgal). We also investigated the expression of protein zonula occuludens-1 (ZO-1) in the epithelial barrier, and barrier function of the irradiated CjECs was evaluated via transepithelial electrical resistance (TEER). Student's t-test was used for statistical analysis.

Results : Expression of p16^INK4a was observed in 6 (60%) of the 10 conjunctival tissue samples, with an average positive rate of 12.3% in each sample, while no p16^INK4a expressing cells was detected in the other 4 tissue samples, and this expression was not sex-dependent. ZO-1 expression was reduced in tissue samples showing p16^INK4a-positivity but was retained in tissue samples where p16^INK4a was undetectable. In irradiated CjECs, there was a significant increase in p16^INK4a expression and SA-βgal activity (and a decrease in ZO-1 expression) both at RNA and protein levels compared to control cells (P<0.05). Moreover, compared with non-irradiated CjECs, TEER was significantly reduced in irradiated CjECs (P<0.05).

Conclusions : We report here the presence of senescent cells in the conjunctival epithelium of elderly subjects, the acquisition of senescent phenotypes in CjECs induced to senescence upon irradiation, and the contribution of ocular senescent cells in the decrease of epithelial barrier function.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.