Abstract Work proposed in the current supplement complements research proposed in the parent NINDS R01 NS108501- 01 “Endothelial-Pericyte Crosstalk in Diabetic Stroke”. Studies in the parent R01 were focused on the acute vascular changes that are common after diabetic stroke (i.e., microvascular no-reflow). The current supplement will investigate the long-term cognitive impairment that follows stroke and that is also more common after diabetic stroke (post-stroke cognitive impairment and dementia, PSCID). The parent project specifically investigated the role of endothelial-pericyte signaling (endothelial-derived epoxyeicosatrienoates (EETs) acting on GPR39 on peri-capillary pericytes) in microvascular no-reflow after diabetic stroke. We tested the hypothesis that EETs/CPR39 crosstalk preserves capillary blood flow in brain after ischemia, and that diabetes reduces microvascular endothelial EETs, leading to pericyte contraction and injury and subsequent capillary occlusion (no-reflow). During the course of the study, we made the observation that in the delayed phase after stroke, pericytes detach from capillaries, proliferate and migrate away from capillaries. We have also observed that these pericytes undergo morphological transformation that is consistent with pericyte activation. Activated pericytes have been reported to play a pro-inflammatory as well anti-inflammatory role under different disease conditions. However, the role of pericyte activation during the chronic phase of recovery from stroke and in post- stroke cognitive impairment under diabetic conditions is not clear. We will test the hypothesis that in normal brain, pericyte activation promotes recovery and functional repair after stroke. Under diabetic conditions, activated pericytes switch to a pro-inflammatory phenotype that contributes to neuroinflammation and neuronal dysfunction underlying PSCID. We propose to conduct single-cell RNAseq (scRNAseq) and single-cell mass cytometry (scCyTOF) of pericytes isolated from male and female, diabetic and no-diabetic NG2-DsRed mice, which label pericytes in red, at 1 day (when pericytes are still attached to capillaries) and 7 days after stroke (when pericytes are activated and detached from capillaries). Single-cell RNAseq will provide new information on pericyte heterogeneity after stroke under diabetic and non-diabetic conditions (on a spectrum ranging from quiescent, capillary attached pericytes to activated, migrating and proliferating pericytes). Furthermore, differential gene expression analysis of pericyte subpopulations from diabetic and non-diabetic brains will provide insight into the role of pericyte heterogeneity in recovery from stroke and post-stroke dementia. We will confirm RNAseq results and further phenotype pericyte subpopulations using scCyTOF.

Project Narrative Having diabetes increases the risk of stroke and worsens its outcome. We will investigate the role of brain cells called pericytes in recovery from stroke and in the development of post-stroke dementia (PSD). Understanding this process may lead to the development of therapies that can prevent and treat PSD.