Abstract DP391: Astrocyte-TLR4 signaling is Sufficient to induce Astrogliosis following Focal Cerebral Ischemia

Astrogliosis is a complex reaction to cell injury, including focal cerebral ischemia. However, the cellular and molecular mechanisms that control this important process are still poorly understood. Without this information, harnessing the full potential of modulating astrogliosis in focal cerebral ischemia cannot be realized. We hypothesized that primary cortical astrocytes with TLR4-deficiency would exhibit a transcriptome with decreased astrocyte reactivity compared to WT astrocytes following oxygen glucose deprivation (OGD). Using a model of in vitro ischemia, OGD, widely used to simulate cerebral ischemia, we characterized the transcriptomic profile of astrocytes with TLR4 deficiency (TLR4-/-) compared to wild type (WT) cortical astrocytes using bulk RNAseq. We extracted total RNA from TLR4-/- and WT primary cortical astrocytes following 12h OGD. RNA was subjected to bulk RNAseq, and we completed bioinformatic analysis on the resulting differentially expressed genes. We also determined astrocyte reactivity, using GFAP immunofluorescence, on coverslips seeded with WT and TLR4-/- astrocytes subjected to 12h OGD. Our results show that astrocytes with TLR4 deficiency express significantly decreased levels of several genes associated with astrocyte reactivity following focal cerebral ischemia, following 12h OGD, compared to WT astrocytes. We also confirmed that, morphologically, astrocytes with TLR4-/- deficiency have significantly less astrocyte reactivity as measured by GFAP immunoreactivity and immunofluorescence compared to WT astrocytes following 12h OGD. GO and KEGG analyses of our results also showed the involvement of the astrocyte-TLR4-/- signaling pathway in novel pathways not previously commonly associated with astrocyte TLR4 signaling, such as synaptogenesis and calcium signaling. These results provide a more in-depth understanding of the role of the TLR4 signaling pathway in astrocyte reactivity following focal cerebral ischemia and identify novel potential functions of astrocyte TLR4 signaling. Importantly, these results suggest that astrocyte-TLR4 signaling, alone, is sufficient to induce astrogliosis following focal cerebral ischemia.