Sanchez, T. | bioRxiv Science

bioRxiv2018

Exacerbation of blood brain barrier dysfunction and brain injury after aneurysmal subarachnoid hemorrhage in endothelial specific S1PR1 knock out mice.

Blood brain barrier (BBB) dysfunction upon ischemia and hypoxia has been implicated in the exacerbation of neuronal injury in stroke. Despite the therapeutic potential of the cerebrovascular endothelium, the limited understanding of the endothelial signaling pathways governing BBB function restricts progress towards developing novel therapeutic approaches specifically targeting the endothelium. Sphingosine-1-phosphate (S1P) is a potent modulator of endothelial function via its receptors (S1PR). Recent human and mouse studies indicate that vasoprotective endothelial S1P signaling via S1PR1 may be impaired in cardiovascular and inflammatory diseases. Herein, we investigated the expression of S1PR1 in the mouse and human cerebrovascular endothelium and the role of endothelial-specific S1PR1 signaling in brain injury in a mouse model of aneurysmal subarachnoid hemorrhage (SAH), the most devastating type of stroke. We found that S1PR1 is the most abundant S1PR transcript in the mouse brain and in mouse and human brain endothelial cells (20-100 mRNA copies per cell). S1PR1 transcripts were significantly enriched (~6 fold) in mouse cortical microvessels compared to total brain. Using the S1PR1-eGFP knock in mouse, we found that S1PR1-eGFP is abundantly expressed in the cerebrovascular endothelium in the mouse brain. A similar pattern of expression was observed in human brain samples. Endothelial specific deletion of S1PR1 in adult mice (S1pr1 flox/floxxCdh5-CreERT2, referred to as S1pr1iECKO), resulted in exacerbation of brain edema, neuronal injury and worsened neurological outcomes upon SAH compared to S1pr1 flox/flox littermates. No differences in the subarachnoid blood, hemostasis or cerebral blood flow changes during and after SAH were found between groups. Mechanistically, S1pr1iECKO exhibited aggravated BBB dysfunction and increased phosphorylation of myosin light chain (MLC) in isolated cortical microvessels, a downstream effector of the Rho-ROCK pathway implicated in endothelial inflammation and barrier dysfunction. Taken together, our data indicate that S1PR1 is an endogenous protective signaling pathway in the endothelium, critical to maintain BBB function and to mitigate neuronal injury in pathological conditions. Thus, the therapeutic and diagnostic potential of the endothelial sphingosine-1-phosphate pathway in stroke deserves further study.

neuroscience↗

bioRxiv2018

An optimized method for the isolation and the molecular characterization of cerebral microvessels in vivo

The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels, with consistent cellular compositions, and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized method for microvessel isolation from mouse brain cortex, which yields microvessel fragments (diameter <50 m, 89.3% 3-5 m) with consistent populations of discrete blood-brain barrier components (endothelial cells, pericytes, and astrocyte end feet), retaining high RNA integrity and protein postranslational modifications (e.g. phosphorylation). We demonstrate that this method allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signalling pathways in mice subjected to drug administration. We also describe the isolation of genomic DNA and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. Therefore, this protocol will be of great use to improve the understanding of the molecular mechanisms governing cerebrovascular dysfunction, which may help the development of novel therapies for stroke and other neurodegenerative diseases.

neuroscience↗