Endothelial-Pericyte Crosstalk in Diabetic Stroke

摘要 本附录中建议的工作是对母NINDS R01 NS108501中建议的研究的补充- 01“糖尿病卒中中的内皮-周细胞串扰”。在亲本R01中的研究集中在急性 糖尿病卒中后常见的血管改变(即微血管无复流)。目前的副刊 将研究中风后的长期认知障碍，糖尿病后更常见的认知障碍 卒中(卒中后认知障碍和痴呆，PSCID)。母项目专门调查了 内皮-周细胞信号转导(内皮源性环氧二十碳三烯酸酯(EETs)对GPR39的作用 糖尿病卒中后微血管无复流。我们测试了这一假设 EETS/CPR39串扰保护脑缺血后毛细血管血流量，减少糖尿病 微血管内皮细胞EETs，导致周细胞收缩和损伤及随后的毛细血管闭塞 (无回流)。在研究过程中，我们观察到在中风后的延迟期， 周细胞从毛细血管中分离、增殖并从毛细血管中迁移。我们还观察到， 这些周细胞经历了与周细胞激活相一致的形态变化。已激活 据报道，周细胞在不同疾病下既发挥促炎作用，又发挥抗炎作用。 条件。然而，周细胞激活在卒中慢性恢复期和卒中后的作用 糖尿病条件下的卒中认知损害尚不清楚。我们将检验这一假设，在正常情况下 大脑，周细胞的激活促进中风后的恢复和功能修复。在糖尿病的情况下， 激活的周细胞转变为促炎表型，从而导致神经炎症和神经元 PSCID的潜在功能障碍。我们建议进行单细胞RNAseq(ScRNAseq)和单细胞质量 从雄性和雌性、糖尿病和非糖尿病NG2-DsRed小鼠分离的周细胞的细胞计数(ScCyTOF)， 在中风后1天(当周细胞仍然附着在毛细血管上)和7天时，用红色标记周细胞 (当周细胞被激活并与毛细血管分离时)。单细胞RNAseq将提供新信息 糖尿病和非糖尿病条件下中风后周细胞异质性的研究(范围从 静止的、毛细血管附着的周细胞与激活的、迁移的和增殖的周细胞)。此外， 糖尿病和非糖尿病脑周细胞亚群的差异基因表达分析将提供 洞察周细胞异质性在卒中和卒中后痴呆康复中的作用。我们会确认 RNAseq结果和使用scCyTOF的进一步表型周细胞亚群。

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.