Hello, my name is Christine Che and I am an NIH StrokeNet Research Fellow at Wake Forest School of Medicine. My presentation is on the microglia activation states as a novel biomarker for outcomes after spontaneous intracerebral hemorrhage. Dr. Wolfe is the Site Co-PI of the NIH StrokeNet Western North Carolina RCC. Otherwise, we have no disclosures. ICH accounts for 20% of all strokes worldwide, about 2 million cases a year. There are approximately 80,000 cases annually in the United States. The 30-day mortality is 40-50% and only 20% of patients reach functional independence by 6 months. To date, there are still no good medical or surgical treatment protocols to treat the primary and secondary brain injury. The onset of ICH causes neuronal and inflammatory cell death, resulting in the release of pro-inflammatory factors. Microglia, the resident macrophages of the CNS, become activated in response to hemorrhage. There are two activation phenotypes, M1 and M2. The M1 phenotype is pro-inflammatory and is activated within minutes of ictus. Their function is a propagation of inflammation with release of inflammatory cytokines, chemokines, and other neurotoxic mediators. The M2 phenotype is anti-inflammatory and begins to appear about 24 hours after ictus. M2 microglia are key in hematoma and debris removal, tissue repair, neurogenesis and angiogenesis, and neuroprotection. We hypothesize that early conversion of microglia from a pro-inflammatory to an anti-inflammatory phenotype correlates with decreased parahematoma edema and improved clinical outcome. Our aims are to isolate microglia in the post-operative drain fluid of ICH surgical patients, determine their activation states via cell surface marker quantification, and correlate activation states with radiographic imaging and clinical outcome. This is a proof-of-concept study of five patients with spontaneous ICH-requiring evacuation within 24 hours of ictus with six-month follow-up. We will utilize single-cell Western blotting, which only needs about 50,000 cells in a sample to identify microglia phenotypes by cell surface markers. After surgical intervention, within the first 72 hours post-ictus, patients will undergo clinical assessments, collection of peripheral blood and post-operative drain fluid, and imaging. All follow-up visits include clinical outcomes measures, collection of peripheral blood, and a CT scan to follow the resolution of hematoma and parahematoma edema. This slide is an example of fluorescence-activated cell sorting on a patient sample, a technique we use to pull microglia from the post-operative drain fluid. The initial run includes all cells found in the fluid, after which we isolated single cells. We use CD45 antibodies to identify leukocytes. Preliminary data suggests that 95% of cells in the post-operative drain fluid are leukocytes. Once hematogens, monocytes, and macrophages have been sorted out, we then isolate a microglia with the use of P2Y12 receptor antibody, which has recently been found to be specific for microglia. Microglia made up 75% of this total leukocyte population. We perform single-cell Western blotting with microglia-specific cell surface markers, TMEM119 and P2Y12. TMEM119 is ubiquitous in all microglia phenotypes. In preclinical studies, downregulation of P2Y12 receptors have been shown to be specific to M2. As you can see here, there are three microglia subsets. At this point, it is unclear if these are different microglia subset lineages or indication of activation states. This peak area of zero only means that the protein was below the set detection limit. In summary, microglia can be isolated from the postoperative drain fluid of ICH patients. Microglia activation phenotypes can be identified by single-cell Western blot technique. This study informs understanding of neuroinflammation after ICH with future goals of identifying therapeutic targets. In the future, we will perform single-cell Western blotting to determine the phosphorylation states of pro-inflammatory transcription factors, STAT1 and STAT3, and anti-inflammatory transcription factors, STAT6, over time. We will use RNA-seq to identify signaling pathways involved in the microglia phenotype shift in the first 72 hours after ICH ictus. And finally, we will utilize DNA methylation to identify pro-inflammatory and anti-inflammatory genes involved in neuroinflammation. These are my references. I would like to thank Drs. Wolff and Asmus for their mentorship, and thank you for your time.

microglia activation states

biomarker

spontaneous intracerebral hemorrhage

ICH

Christine Che