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Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH)

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Abstract

Activation of innate immunity contributes to secondary brain injury after experimental subarachnoid hemorrhage (eSAH). Microglia accumulation and activation within the brain has recently been shown to induce neuronal cell death after eSAH. In isolated mouse brain capillaries after eSAH, we show a significantly increased gene expression for intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Hence, we hypothesized that extracerebral intravascular inflammatory processes might initiate the previously reported microglia accumulation within the brain tissue. We therefore induced eSAH in knockout mice for ICAM-1 (ICAM-1−/−) and P-selectin glycoprotein ligand-1 (PSGL-1−/−) to find a significant decrease in neutrophil-endothelial interaction within the first 7 days after the bleeding in a chronic cranial window model. This inhibition of neutrophil recruitment to the endothelium results in significantly ameliorated microglia accumulation and neuronal cell death in knockout animals in comparison to controls. Our results suggest an outside-in activation of the CNS innate immune system at the vessel/brain interface following eSAH. Microglia cells, as part of the brain’s innate immune system, are triggered by an inflammatory reaction in the microvasculature after eSAH, thus contributing to neuronal cell death. This finding offers a whole range of new research targets, as well as possible therapy options for patients suffering from eSAH.

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Acknowledgments

We thank Dr. Urban Deutsch (Theodor Kocher Institute, Bern, Switzerland) for maintaining the breeding stock of the PSGL-1−/− and ICAM-1−/− C67BL/6 mice. We thank Lars Winkler (FMP Berlin Buch) for the preparation of isolated brain capillaries. We thank Dr. Susan Brandenburg for FACS analyses.

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    Authors and Affiliations

    1. Experimental Neurosurgery, Charité–Universitätsmedizin Berlin, 10117, Berlin, Germany

      Etienne Atangana, Ulf C. Schneider, Kinga Blecharz, Salima Magrini, Josephin Wagner, Melina Nieminen-Kelhä, Irina Kremenetskaia & Peter Vajkoczy

    2. Department of Anaesthesia and Intensive Care Medicine, Universitätsmedizin Göttingen, 37099, Göttingen, Germany

      Etienne Atangana

    3. Department of Neurosurgery, Charité–Universitätsmedizin Berlin, 12200, Berlin, Germany

      Ulf C. Schneider & Peter Vajkoczy

    4. Department of Neuropathology, Charité–Universitätsmedizin Berlin, 10117, Berlin, Germany

      Frank L. Heppner

    5. Theodor Kocher Institut, Universität Bern, 3012, Bern, Switzerland

      Britta Engelhardt

    6. Department of Neurosurgery, Charité–Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

      Peter Vajkoczy

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    Correspondence to Peter Vajkoczy.

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    This work was supported by the Deutsche Forschungsgemeinschaft (SFB TRR 43 and NeuroCure Exc 257 to FLH and PV, as well as HE 3130/6-1 to FLH) and the Federal Ministry of Education and Research (DLR/BMBF; Kompetenznetz Degenerative Demenzen) to FLH and the Swiss Heart Foundation to BE.

    Conflict of Interest

    Etienne Atangana has no conflict of interest.

    Ulf C. Schneider has no conflict of interest.

    Kinga Blecharz has no conflict of interest.

    Salima Magrini has no conflict of interest.

    Josephin Wagner has no conflict of interest.

    Melina Nieminen-Kelhä has no conflict of interest.

    Irina Kremenetskaia has no conflict of interest.

    Frank Heppner has no conflict of interest.

    Britta Engelhardt has no conflict of interest.

    Peter Vajkoczy has no conflict of interest.

    Ethical Approval

    All applicable international, national, and institutional guidelines for the care and use of animals were followed.

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    Etienne Atangana and Ulf C. Schneider contributed equally to this work.

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    Fig. S1

    Illustration of the purity of isolated brain vessels by co-staining with Zonula occludens-1 (ZO-1) and claudin-5, both being specific vascular tight junction markers. Cell bodies were counterstained with DAPI. (PPTX 145 kb)

    Fig. S2

    Representative dot plots of the FACS analysis gated for CD45 and CD11b to detect an up-regulation of lymphocytes in brain homogenates as a supplement to the data, summarized in Fig. 5. No significant differences in lymphocyte numbers were found in relation to all living cells (a, upper), or in relation to all living immune cells (a, lower). The gating strategy is displayed (b) (PPTX 78 kb)

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    Atangana, E., Schneider, U.C., Blecharz, K. et al. Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH). Transl. Stroke Res. 8, 144–156 (2017). https://doi.org/10.1007/s12975-016-0485-3

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