Abstract WP331: Diverse Microglia Morphologies Induced By Ischemic Stroke And Reperfusion Are Not Accompanied By Altered Brain Inducible Nitric Oxide Synthase Expression.

Abstract

Microglia cells continuously survey the healthy brain in a ramified morphology and, in response to injury, undergo progressive morphological and functional changes that encompass microglia activation. Activated microglia may contribute towards secondary injury during ischemic stroke and reperfusion (ISR) through pro-inflammatory mechanisms and increased oxidative stress. Nitric oxide (NO), a product of microglia inducible NO synthase (iNOS) expression, contributes toward increased oxidative stress after brain injury. Although ideally positioned for an immediate response, microglia morphological responses have not been characterized after ISR. In addition it is not well understood if microglia morphology correlate to microglia function relative to oxidative stress. Our aim was to quantify microglia morphology and iNOS expression, an indicator of NO production, during the evolution of cerebral injury after ISR. Anesthetized mice were subjected to focal ischemic stroke accompanied by varied lengths of reperfusion (ischemia only, 8hr and 24hr reperfusion). Microglia process length/cell and number of endpoints/cell was quantified from immunofluorescent confocal images of brain regions spatially related to the necrotic core using a skeleton analysis method developed for this study. Live cell microglia morphology and process movement was measured from movies acquired in acute brain slices from GFP-CX3CR1 transgenic mice after IS and 24hr reperfusion. iNOS expression was measured at all time-points and similar brain regions using western blot techniques. We show a significant spatiotemporal relationship between microglia process length/cell and number of process endpoints/cell and evolving brain lesion after ISR (F=4.17, p<0.001, F=3.57, p<0.01). After 24hr of reperfusion, process activity was significantly decreased the peri-infarct region vs. contralateral (p<0.05). However, iNOS protein expression remained unchanged in all ipsilateral regions vs. matching contralateral regions after 60min ischemia and first 24hr of reperfusion. While microglia morphological responses to ISR are immediate and diverse, we have yet to confirm microglial sources of NO are increased within the first 24hr of ISR.