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Utilizing Multi‐Color Flow Cytometry and FACS to Simultaneously Examine Effects of Intermittent Hypoxia on Microglia, Neurons, and Astrocytes

Intermittent Hypoxia (IH) as is experienced during sleep apnea causes significant cortical and hippocampal neuronal apoptosis correlating with a decline in cognition. It is well‐established that neuro‐inflammation underlies much of this neuronal loss, but little is known about the contributing CNS cell types. In the present studies, we exposed rodents to a paradigm of intermittent hypoxia consisting of 8 hours of intermittent hypoxia (2 min 21% O2, 2 min 10.5% O2) or normoxia (21% O2) followed by a 16 hour reoxygenation at room air for either 1, 3, 7, 14, or 28 days. We have developed a multi‐color flow cytometry panel that allows simultaneous examination of protein levels from three distinct CNS cell populations (microglia‐CD11b+, astrocytes‐GFAP+, and neurons‐Neurofilament+) in the cortex and hippocampus, regions of highest neuronal loss after IH. We investigated changes in pro‐inflammatory (TNFα, IL‐1β, iNOS, and COX‐2) and trophic (VEGF, BDNF, IGF‐1) factors. In addition, we utilized markers of apoptosis (Caspase‐3) and proliferation (Ki‐67) to assess neuronal loss and glial proliferation. After this analysis, we utilized Fluorescence‐Activated Cell Sorting (FACS) to sort pure populations of these 3 cell types for gene expression analysis by qRT‐PCR. This powerful technique enables examination of simultaneous, multi‐cellular contributions to IH‐induced neuropathology.