Purpose: Ischemia and reperfusion (I/R) injury damages retinal neurons. Retinal injury is accompanied by activation of microglia, which scavenge the dead or dying neurons, but increasing evidence now indicates that amoeboid-shaped microglia cells activated in the brain after ischemia have neurotoxic and damaging properties in their own right. A previous study showed that postconditioning with carbon monoxide (CO) protects retinal ganglion cells (RGCs) after I/R through anti-apoptotic and anti-inflammatory mechanisms. The present study was designed to investigate and quantify the activation of retinal microglia after I/R with and without CO postconditioning.
Methods: Adult Sprague-Dawley rats underwent retinal ischemia by increasing the ocular pressure to 120 mmHg for 1 h through a needle inserted into the anterior chamber. Reperfusion was induced by removing the needle. After I/R, one group of animals was kept in a CO (250 ppm) atmosphere for 1 h; the other group was kept in room air (Air). At 1, 2, 3, and 7 days after I/R, the eyes were enucleated and fixed. Intracardiac blood was analyzed for systemic effects of CO or I/R. Retinal cross sections were taken from the middle third of the eye and were stained with anti-Iba-1. Microglia cells were graded as amoeboid or ramified phenotypes according to morphologic criteria. Retinal thicknesses were determined.
Results: Evaluation of retinal tissue revealed a significant reduction of amoeboid microglia cells after I/R + CO when compared to the I/R + Air group. The peak number of amoeboid microglia was observed at day 2 post-I/R + Air. This rise was attenuated by CO postconditioning (815 versus 572 cells/mm2 for I/R + Air versus I/R + CO, respectively; p = 0.005). CO reduced and further postponed the peak in the numbers of amoeboid and ramified microglia cells in ischemic eyes and prevented microglial activation in the contralateral eyes. I/R-induced leucocytosis was inhibited by CO inhalation. The reduction of retinal thickness after I/R was more serious after Air inhalation when compared to the CO group.
Conclusions: Numerous activated microglia cells appear in the inner retina after I/R, and CO-treatment significantly attenuates this glial response. Antagonism of microglial activation may be a further neuroprotective effect of CO, apart from its direct anti-apoptotic capacity.
Keywords: Ischemia; Microglia; Neuroinflammation; Neuroprotection; Retina; Retinal ganglion cell.