Abstract
Purpose :
Histatin-1 belongs to a family of low molecular weight salivary and lacrimal peptides that have diverse wound healing, anti-inflammatory and antimicrobial properties. Topically administered Histatin-1 has been shown to speed-up healing of the epithelium following anterior keratectomy in rabbits and to also preserve and restore stromal keratocytes and corneal endothelium (Burke, et al., ARVO 2022). This study examined viability of corneal endothelial cells in ex-vivo human cornea buttons stored at room temperature instead of standard hypothermic condition to accelerate injury to endothelial cells.
Methods :
Eleven pairs of ex-vivo human corneal buttons of research-consented donors (F=7, M=4) were used in this study. Median age and corneal endothelial cell density (CECD) were 64 years and 2119 cells/mm2, respectively. Corneas were stored in FDA-approved corneal storage media (life 4C media, Optisol, and Eusol C) at 4 °C in 20 mL storage chambers for 3 days then at room temperature (24-26 °C) for the rest of the 2-week experiment. For each corneal pair, one was dosed with 200 µL of Histatin-1 formulation three times per week to maintain a concentration of 1 µM; the other mate cornea was dosed with PBS as control. Dosing started on Day 1 of hypothermic storage conditions. The corneal endothelial layer was evaluated for cell non-viability by imaging the central 80% of the cornea stained with trypan blue (n=8) and evaluated for apoptotic cells by imaging the center region after staining with Annexin-V (n=3). Annexin images were acquired using 20x objective (0.14 mm2) on Leica DMi8 epifluorescence microscope. Data are expressed as percent and mean ± SEM area measurements.
Results :
The area of non-viable endothelium by trypan blue staining was 1.8-fold greater in the control group (24 ± 6%) than in the Histatin-1 group (13 ± 3%). This reduction in cell viability was associated with a nearly 8-fold increase in Annexin-V staining in the control group compared to the Histatin-treated corneas (4073 ± 1623 µm2 v/s 514 ± 164 µm2, respectively).
Conclusions :
Histatin-1 markedly decreases human corneal endothelial cell loss under ex-vivo, stressed temperature conditions. This is associated with a substantial reduction in biomarkers of apoptosis. These results may have implications for corneal preservation and for treating various diseases of the cornea.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.