Dietary restriction mitigates age-related accumulation of DNA damage, but not all changes in mouse corneal epithelium

The cornea protects the anterior eye and accounts for two thirds of the eyes refractive capacity. The homeostasis of corneal epithelium is thought to be maintained by putative stem cells residing in the epithelial basal layer. As a tissue constantly exposed to environmental stress, the cornea is hypothesised to accumulate persistent DNA damage events with time in stem cell populations. Recently, telomere associated DNA damage foci (TAFs) have been suggested as a marker for persistent DNA damage which can be used to detect senescent cells during ageing. Dietary restriction (DR) is the only known non-genetic intervention that is able to increase both life and health span among various animal species. The aim of this study was to analyse changes in corneal properties with age and under 16 months of DR. We employed immunofluorescence staining for ɣH2A.X together with telomere fluorescence in situ hybridisation (immuno-FISH) on mouse corneas from young, old ad libitum (AL) fed as well as dietary restricted (DR) mice. Our data show that the central corneas of old mice had significantly more general and telomere-associated DNA damage compared to young mice while DR treatment was able to reduce the amount of DNA damage significantly. We also found that the thickness of the peripheral region of the cornea, where the putative stem cells may reside, decreased with age regardless of whether the animals underwent DR treatment or not. Number of bullae, which indicates corneal edema, accumulated in old corneas in the central area and DR treatment mitigated the formation of these bullae. Moreover, the protein levels of the stem cell marker TAp63 decreased with age only in the central but not the peripheral region of the cornea. This finding suggests that epithelial progenitors might be better maintained in the peripheral than the central cornea during ageing. Together with the finding that the peripheral corneal showed no increase in DNA damage during age, we speculate that in mice, like humans, the putative stem cells reside in the peripheral cornea.