Restoration of Blood-Retinal Barrier Integrity Prevents Neurodegeneration in Glaucoma

Abstract

Purpose : Vascular dysfunction is a major component of glaucoma pathophysiology. The contribution of blood-retinal barrier (BRB) disruption during glaucomatous neurodegeneration is poorly understood. Here, we tested the hypotheses that: i) BRB dysfunction occurs in the early stages of glaucomatous damage, and ii) rescue of barrier integrity promotes retinal ganglion cell (RGC) survival.

Methods : Ocular hypertension (OHT) was induced by intracameral injection of magnetic microbeads in C57BL/6 mice. Fundus fluorescein angiography was used to longitudinally image vascular leakage into the retinal parenchyma. Histological analysis of BRB permeability was performed by stereological sampling of whole-retina or peripapillary areas after retro-orbital administration of fluorescent dextran (3K, 10K, 70K MW). Two weeks post-OHT (OHT-2w), dextran fluorescence was quantified using confocal microscopy and ImageJ software. Endothelial cell tight junction proteins were quantified by immunoblotting. Endothelium-specific gene supplementation of Claudin-5 was achieved by intravenous delivery of recombinant adeno-associated virus (AAV.Cldn5). RGC density was quantified in RBPMS-stained retinas using a stereological approach.

Results : Fundus angiography showed a progressive increase in fluorescein leakage starting at OHT-1w, which remained elevated thereafter (n=4 mice/group). Whole-retina and peripapillary imaging at OHT-2w, when OHT is stable but no significant RGC loss is detected, showed a substantial increase in dextran leakage in all vascular plexuses of the retina (t-test p<0.01, n=8 mice/group). A screening of endothelial tight junctions revealed reduced Claudin-5 expression in eyes with OHT relative to sham controls (t-test p<0.01, n=6 mice/group). Selective AAV-mediated Cldn5 gene transfer to endothelial cells reduced fluorescein leakage (t-test p<0.01, n=5 mice/group) and improved RGC survival (OHT-3w) compared to eyes treated with a control virus (t-test p<0.001, n=5 mice/group).

Conclusions : Our data demonstrate early disruption of BRB integrity during OHT-induced damage. We show that Cldn5 gene transfer to endothelial cells effectively reduces vascular leakage and improves RGC survival in glaucomatous eyes. These findings suggest that strategies that restore vascular integrity are beneficial to counter neurodegeneration in glaucoma.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.