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.