Abstract
Purpose :
Tight regulation of blood supply is critical for maintaining retinal homeostasis, however, the underlying mechanisms are largely unknown. Here, we examined how microglia contribute to vasoregulation in the retina
Methods :
Live cell imaging was used to assess dynamic control of vessel calibre by microglia in retinal explants from Cx3cr1-GCaMP3 mice which express a genetically encoded Ca2+ indicator in microglia. Pharmacological agents used included ATP, the P2 antagonist PPADS, the mGluR agonist trans-ACPD, and fractalkine, the sole ligand for Cx3cr1. In vivo assessment of vessel calibre and blood flow was performed in C57BL/6J mice, and Cx3cr1+/GFP and Cx3cr1GFP/GFP mice using optical coherence tomography angiography and fluorescein angiography. Microglia from C57BL/6 and Cx3cr1GFP/GFP mice were also assessed for expression of vasoregulatory genes using RNAseq.
Results :
Frequent spontaneous Ca2+ transients were observed in microglia which regularly (>60%) led to constriction of neighbouring capillaries. The magnitude of constriction was significantly correlated with the change in intracellular Ca2+ (n=253 events from 9 animals). These responses were significantly enhanced by application of exogenous ATP (n=3, p<0.005). Application of fractalkine also induced vasoconstriction but in the absence of microglial Ca2+ (n=3-4, p<0.05). In vivo imaging revealed loss of Cx3cr1 led to dilation of retinal capillaries (n=4-6, p<0.05) and altered blood flow (n=9-12, p<0.05). RNAseq data showed a significant downregulation of angiotensin converting enzyme (Ace; -9.5 fold change, n=5-6, p=9.9E-13), suggesting Cx3cr1 may regulate vessel calibre via the renin angiotensin system (RAS). This was confirmed by using a RAS inhibitor, which prevented fractalkine-induced vasoconstriction in retinal explants (n=9, p<0.05).
Conclusions :
We have shown that retinal microglia can regulate vessel calibre via a Ca2+-dependent pathway mediated by purinergic and glutamatergic signalling, and a Ca2+-independent pathway mediated by Cx3cr1 and the RAS. These findings suggest microglia could be important for controlling retinal blood flow and may contribute to a greater understanding of CNS diseases that involve dysregulated blood supply.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.