Blood–brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database (https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

血脑屏障 (BBB) 功能障碍以 BBB 连接蛋白的降解和通透性增加为特征，是急性缺血性中风的重要病理生理学特征。多种神经血管单元 (NVU) 细胞类型的失调与缺血性脑卒中的 BBB 分解有关，再灌注治疗可能会进一步加剧。因此，急性缺血性中风中失调的 NVU 细胞类型的治疗性共同靶向构成了保持 BBB 功能和改善临床结果的有前途的策略。然而，缺乏从同一患病的中枢神经系统 (CNS) 组织中同时分离多种 NVU 细胞类型的方法，这对于识别失调的 NVU 细胞中的治疗靶标至关重要。在这里，我们介绍了 EPAM-ia 方法，这有助于同时分离和分析主要 NVU 细胞类型（内皮细胞、周细胞、星形胶质细胞和小胶质细胞），以识别失调的 NVU 细胞中的治疗靶点以改善 BBB 功能。应用这种方法，我们从小鼠缺血性脑组织中获得了高产量的纯 NVU 细胞，并生成了一个有价值的 NVU 转录组数据库（https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke）。NVU 转录组的解剖显示 de/SGD_Stroke). NVU 转录组的解剖显示 de/SGD_Stroke). NVU 转录组的解剖显示Spp1，编码骨桥蛋白，在缺血性中风 24 小时后在所有 NVU 细胞中高度上调。通过免疫染色证实了中风患者骨桥蛋白的上调，这与小鼠中的相当。通过在小鼠缺血性中风后皮下注射抗骨桥蛋白抗体进行治疗靶向导致所研究的 NVU 细胞类型中骨桥蛋白表达的中和。除了神经胶质细胞活化减弱外，骨桥蛋白中和还与 BBB 保存以及脑水肿减少和出血性转化风险降低相关，从而改善了神经学结果和存活率。这得到了体外骨桥蛋白 BBB 损伤作用的支持。