A novel femtomolar hemodynamic modulation strategy reveals major microvascular defects in glaucoma at single-pericyte scale

Abstract

Purpose : Reduced blood flow and neurovascular deficits are recognized features of glaucoma, but the mechanisms underlying these alterations are poorly understood. Pericytes, the contractile cells that wrap around capillaries, regulate blood supply by modulating capillary diameter. Here, we developed a novel live imaging and femto-scale delivery system to study the role of single-pericyte hemodynamics in glaucoma.

Methods : Ocular hypertension (OHT) was induced by intracameral injection of magnetic microbeads in NG2-DsRed mice, which allow visualization of retinal pericytes. Two-photon laser scanning microscopy (TPLSM) was combined with a femtomolar delivery system (FemtoJet Microinjector) to visualize and modulate single-pericyte longitudinal responses in living mice. A micromanipulator was used to place the microneedle adjacent to a pericyte-capillary pair, and vasomodulators (e.g. ET-1:endothelin-1, NO donors) were delivered at a femtomolar scale. Capillary diameter was measured by placing a linear probe perpendicular to the plane of the vessel, and blood flow was quantified by counting the number of red blood cells crossing a pre-fixed vessel location.

Results : TPLSM adapted with a femto-microinjector provided high spatial-temporal resolution of single-pericyte-capillary responses without affecting neighboring vessels. Our data show decreased capillary diameter and blood flow at pericyte locations in glaucoma (sham: n=13 capillaries, OHT-2 wks: n=18 capillaries, N=5-7 mice/group, p<0.01). Femtomolar delivery of ET-1 in glaucomatous mice exacerbated the magnitude and duration of capillary constriction at pericyte locations and decreased blood flow relative to controls (vehicle: n=11 capillaries, ET-1: n=16 capillaries, N=3-5 mice/group, p<0.001). In contrast, NO donor administration rescued the ability of capillaries to dilate at pericyte locations enhancing blood flow despite OHT (vehicle: n=16 capillaries, NO donor: n=7 capillaries, N=3-5 mice/group, p<0.01), suggesting that microvascular defects in glaucoma are reversible.

Conclusions : We demonstrate the utility of combining TPLSM live longitudinal imaging and femtomolar delivery of vasoactive substances to study neurovascular defects caused by OHT. Our study identifies pericytes as critical regulators of capillary hemodynamics and unveils their potential as therapeutic targets to restore neurovascular function in glaucoma.

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