Abstract
The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal, and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vessel-enriched and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in tight junction protein expression, serum albumin signals and astrocyte activation, i.e., increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.
Highlights
The cerebral vasculature has unique properties since the transfer of molecules to the brain parenchyma is tightly regulated by the blood-brain barrier (BBB) [1,2]
Published mouse brain vessels purification protocols have been used for subsequent immunofluorescence stainings and Western blotting, these studies usually focused on the purified vessels alone, without comparison to the remaining brain parenchyma, or using total brain homogenate for comparison
We evaluated a modified mouse brain vessel-parenchyma fractionation method that permits quantitative protein analyses of the cerebrovascular niche and the vessel-depleted parenchyma fraction and enables investigations requiring analysis of small tissue volumes, e.g., the direct comparison of the two brain hemispheres from the same animal
Summary
The cerebral vasculature has unique properties since the transfer of molecules to the brain parenchyma is tightly regulated by the blood-brain barrier (BBB) [1,2]. Studies have shown involvement of BBB dysfunctions and cerebral blood flow deficits in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [3,6,7]. Disruption of the BBB can lead to secondary damage such as brain edema or hemorrhagic transformation, and to the infiltration of immune cells [8]. Investigations of brain vasculature in such disease conditions require in vivo studies because of the complexity of disease pathologies and the variety of cell types involved. Research relies in part on animal models, and quantitative methods are needed in order to elaborate on the precise mechanisms behind pathology, which in turn eventually leads to the development of new therapeutic strategies
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