Differential regulation of cerebral microvascular transcription by single and repetitive hypoxic conditioning

Systemic conditioning therapeutics afford brain protection at all levels of organization, occurring autonomously for neurons, glia, vascular smooth muscle, and endothelium, which are mediated systemically for the adaptive and innate immune system. The present study was undertaken to examine acute (3 h) and delayed (2 days) gene expression changes in mouse cerebral microvessels following single hypoxic conditioning (HX1) and repetitive hypoxic conditioning (HX9), the latter for which we showed previously to extend focal stroke tolerance from days to months. Microarray (Illumina) analyses were performed on microvessel-enriched fractions of adult mouse brain obtained from the following five groups (naïve; HX1-3h; HX1-2days; HX9-3h; HX9-2days). Differentially expressed genes were analyzed bioinformatically using Ingenuity Pathway Analysis software, with qPCR validating selected up- and down-regulated genes. As expected, some differentially expressed genes were common to more than one treatment or time point, whereas others were unique to treatment or time point. Bioinformatic analyses provided insights into acute (3h) inflammatory and immune signaling pathways that may be differentially activated by HX1 and HX9, with anti-inflammatory and trophic pathways coincident with the ischemia-tolerant phenotype two days after HX1. Interestingly, two days after HX9, microvessels were transcriptionally silent, with only five genes remaining differentially expressed relative to naïve mice. Our microarray findings and bioinformatic analyses suggest that cerebral microvessels from HX1-treated mice exhibit early activation of immune system signaling that is largely suppressed in microvessels from HX9-treated mice. These and other differences between these responses require further study, including at the proteomic level, and with pharmacologic and genetic experiments designed to reveal causality, to reveal further insights into the mechanisms underlying long-lasting stroke tolerance.