Presentation description

The focus of my summer project is to optimize histological procedures to test the hypothesis that intrinsic cytoprotection afforded by endothelial cell (EC) autophagy in response to acute ischemic stroke (AIS) is blunted by aging. First, we substantiated earlier reports that AIS outcomes are worse in older vs. adult mice. Sixty-min transient middle cerebral artery occlusion (tMCAO) reduced perfusion similarly in both groups, but reperfusion (R) at 23-h was greater (p<0.05) in adult vs. older mice. Further, AIS-induced neurobehavioral and motor deficits were more severe (p<0.05) in older vs. adult mice. A second cohort of adult and older mice completed tMCAO + R procedures. At 24 hours, mice completed performance testing, followed by anesthesia, perfusion fixation, and brain extraction. From each of the 20 brains, a coronal 50 µM section from the cerebral cortex of the ipsilesional (ischemic) hemisphere was stained using immunofluorescent antibodies recognizing autophagy-related gene 3 (Atg3), cluster of differentiation 31 (CD31; endothelial cell marker), glucose transporter type 1 (GLUT1; glucose transport marker), neuronal nuclei (NeuN; neuronal marker), ionized calcium-binding adapter molecule 1 (IBA-1; microglial marker), and glial fibrillary acidic protein (GFAP; astrocyte marker). From each section, intensity of the respective immunofluorescent antibody (e.g., GLUT1) colocalizing with the cell of interest (e.g., CD31) was captured from 3 images x 10 regions of interest (ROI) using a Leica DM6000 fluorescence microscope. The mean fluorescence intensity of the 30 ROIs was averaged, and compared among groups using a 2-way ANOVA. To date, we find AIS-induced colocalization of GLUT1 with CD31 is blunted (p<0.05) in ipsilesional hemispheres from older vs. adult mice. Analyses of the remaining endpoints is ongoing. We endeavor to provide insight regarding how aging impacts stroke pathology.