Abstract
Purpose :
Retinal microglial cells contribute to the pathogenesis of several retinal disorders, but the signals that recruit microglia to the site of injury and trigger activation remain unclear. In the brain, extracellular ATP can activate microglial cells and act as a powerful chemoattractant. Our previous work shows ATP is released from astrocytes in the optic nerve head in response to mechanical stretch or increased IOP. This study examines the role of extracellular ATP on the responses by retinal microglia and asks whether purinergic signaling could link strained astrocytes to activated microglia.
Methods :
Primary cultures of microglia were isolated from the mouse retina. Microglial migration was determined with a Boyden chamber. Gene levels were determined with standard qPCR. IOP was elevated to 60 mmHg for 4 hrs using the CEI procedure in mice. IBA1 was visualized using immunohistochemistry.
Results :
ATP was a strong chemoattractant for retinal microglial cells. The number of microglial cells migrating through the Boyden chamber pores doubled when ATP was included in the target wells. This enhanced migration was found both by determining total fluorescence from Hoechst-labeled microglial cells and by counting individual cell numbers. The P2Y12 antagonist AR-C69931 reduced microglial numbers passing through the chamber, implicating the P2Y12 receptor in the response. Exposure of isolated rat retinal microglial cells to 3 mM ATP for 3 hrs increased expression of classical activation marker iNOS and alternative activation marker chi313. In vivo trials suggested increased staining for IBA1 across the retina in response to elevated IOP. In isolated retinal wholemounts from CX3CR1-GFP mice, incubation with the P2X7 receptor agonist BzATP increased the numbers of IBA1-positive cells throughout the retina and around the optic nerve head region.
Conclusions :
ATP acts as a strong chemoattractant for retinal microglial cells and activates the cells. Given that optic nerve head astrocytes release ATP through pannexin hemichannels when stretched, these results implicate the purinergic system in signaling from stressed astrocytes to activated microglial cells in the compromised retina.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.