Glial cells regulate pericyte-driven capillary deficits in glaucoma

Abstract

Purpose : Neurovascular coupling (NVC) is a physiological mechanism that matches blood flow with neuronal activity to bring oxygen and nutrients to active neurons. We recently demonstrated that pericytes regulate NVC in the retina and that pericyte dysfunction leads to reduced blood flow during ocular hypertension (OHT). Here, we asked whether glial cells in the retina and optic nerve contribute to pericyte responses by probing the mechanisms underlying capillary deficits.

Methods : OHT was induced by intracameral injection of magnetic microbeads in reporter mice to visualize pericytes and/or glial cells as well as calcium (Ca²⁺) transients (GCaMP6f). Two-photon laser scanning microscopy (TPLSM) was combined with a femtomolar delivery system for longitudinal trans-scleral imaging of glial-pericyte-capillary responses in living mice. A set of complementary techniques was used to probe cell-specific molecular changes including sc-RNAseq, qRT-PCR, and immunohistochemistry (IHC).

Results : We found a substantial reduction of capillary blood flow at pericyte locations concomitant with intrapericyte Ca²⁺ increase in eyes subjected to OHT relative to sham controls (N=9 mice/group, n= 200-376 capillaries, Student’s t-test, p<0.001). Single cell (sc)-RNAseq revealed changes in Ca²⁺ homeostasis pathways including upregulation of transcripts encoding the Ca²⁺ binding protein S100B in glial cells. IHC confirmed S100B upregulation in Müller cells and astrocytes in the retina and optic nerve head. Delivery of recombinant S100B protein exacerbated intrapericyte Ca²⁺ influx and blood flow impairment (N=11 mice/group, n= 62-76 capillaries, Student’s t-test, p<0.001). In contrast, administration of S100B function blocking antibodies (FBA) decreased Ca²⁺ in pericytes and improved capillary blood flow during OHT stress (blood flow - sham: 100%, n=128 capillaries OHT + control antibody: 65 ± 19%, n=153 capillaries; OHT + FBA: 97 ± 5%, n=576 capillaries, N=4-9 mice/group, ANOVA, p<0.001). Remarkably, S100B neutralization protected RGCs from OHT-induced death (% survival – sham: 100%, OHT + FBA: 93%, OHT + control: 65%, N=11-13 mice/group, ANOVA, p<0.001).

Conclusions : Our data reveal a novel role of glial cells in the regulation of pericyte responses to OHT stress and identify S100B as a critical glia-derived player in NVC deficits.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.