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Characterization of the blood-brain barrier in genetically diverse laboratory mouse strains

ABSTRACT: Genetic variation in the population has an influence on the manifestation of monogenic as well as multifactorial disorders, with the underlying genetic contribution dependent on several interacting variants. Common laboratory mouse strains used for modelling human disease lack the genetic variability of the human population. Therefore, outcomes of rodent studies show limited correlation with human disease. The functionality of brain vasculature is an important modifier of brain diseases. Importantly, the restrictive interface between blood and brain – the blood-brain barrier (BBB) serves as a major obstacle for CNS drug delivery. Using genetically diverse mouse strains, we aimed to investigate the phenotypic and transcriptomic variation of the intact BBB in different inbred mouse strains.We investigated the heterogeneity of brain vasculature in recently wild-derived mouse strains (CAST/EiJ, WSB/EiJ, PWK/PhJ) and long-inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, DBA/2J, NOD/ShiLtJ) using different phenotypic arms. We used immunohistochemistry and confocal laser microscopy followed by quantitative image analysis to determine vascular density and pericyte coverage in two brain regions – cortex and hippocampus. Using a low molecular weight fluorescence tracer, sodium fluorescein and spectrophotometry analysis, we assessed BBB permeability in young and aged mice of selected strains. For further phenotypic characterization of endothelial cells in inbred mouse strains, we performed bulk RNA sequencing of sorted endothelial cells isolated from cortex and hippocampus. We did not detect differences in cortical and hippocampal vessel density and pericyte coverage among all the investigated strains. The staining patterns of endothelial arteriovenous zonation markers were similar in different strains. BBB permeability to a small fluorescent tracer, sodium fluorescein, was also similar in different strains. Transcriptomic analysis of endothelial cells revealed that sex of the animal was a major determinant of gene expression differences. In addition, the expression level of several genes implicated in endothelial function and BBB biology differed between wild-derived and long-inbred mouse strains. Our analysis shows that although there were no major differences in parenchymal vascular morphology and BBB permeability between investigated mouse strains or sexes, transcriptomic differences of brain endothelial cells point to variation in gene expression of the intact BBB. These baseline variances might be confounding factors in pathological conditions that may lead to a differential functional outcome dependent on the sex or genetic polymorphism.

ORGANISM(S): Mus musculus

PROVIDER: GSE173793 | GEO | 2021/08/03

REPOSITORIES: GEO

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Project description:The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB. The brain microvascular fragments were isolated from mice with different genotypes, each represented by 3-4 biological replicates. Genotypes 1-2: Platelet derived growth factor-B (PDGF-B) retention-motif knockout (pdgfbret/ret) represent the pericyte-deficient situation, and the heterozygous mice (pdgfbret/+) are used as controls. Genotypes 3-4: Hypomorphic PDGF-B mutants that rescue pdgfb-/- null mice, in which a one copy of a conditionally silent human PDGF-B transgene targeted to the Rosa 26 locus (R26P) is turned on by endothelial-specific expression of Cre recombinase. In this data set these mice are named as Tie2Cre, R26P+/0, pdgfb-/- (representing the pericyte-deficient situation). Mice wt for pdgfb (pdgfb+/+) and carrying one silent copy of R26P (R26P+/0), are used as controls. Genotype 5: Adult Notch3+/+ wildtype (WT).

Project description:Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae that leads to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from hematogenous source into the brain across the blood-brain barrier (BBB); the mechanisms however are still poorly understood. Current treatment strategies include adjuvant dexamethasone therapy for cerebral edema, the prime reason for neurological complications and mortality, which is followed up by antibiotics. The success of corticosteroids is however inconclusive, necessitating the development of new therapies for controlling cerebral edema. We have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we therefore hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is critically involved in the transmigration of pathogens across the BBB. In human and murine meningitis brain samples, HIF-1 activation was observed by immunohistochemistry. HIF-1α/VEGF expression and permeability was therefore analyzed in vitro in infected brain endothelial cells (ECs). Localization of S. pneumoniae in brain ECs from mouse/human sources was visualized in vitro and in vivo by confocal, super-resolution, and electron microscopy. S. pneumoniae infection in brain ECs resulted in upregulation of HIF-1α/VEGF by Western blotting and qRT-PCR that was associated with increased paracellular permeability, which was supported by bacterial localization at cell-cell junctions. RNA sequencing of brain microvessels from hematogenously infected mice with increased permeability and S. pneumoniae deposition in the brain showed upregulation of genes in HIF-1α/VEGF pathway. Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells and by using primary brain ECs from HIF-1α KO mice revealed a reduced endothelial permeability and transmigration of S. pneumoniae. We thus demonstrate a critical role for HIF-1α/VEGF in transmigration of S. pneumoniae across the BBB and propose targeting this pathway to prevent BBB dysfunction in infections.

Project description:Rationale. The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/b-catenin signaling is responsible for the early phases of brain vascularization and blood-brain barrier differentiation. However, this signal declines after birth and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown. Objective. Sox17 constitutes a major downstream target of Wnt/b-catenin in endothelial cells and regulates arterial differentiation. In the present paper, we asked whether Sox17 may act downstream of Wnt/b-catenin in inducing BBB differentiation and maintenance. Methods and Results. Using reporter mice and nuclear staining of Sox17 and b-catenin, we report that while b-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo, and progressively declines after birth. In search of Sox17 mechanism of action, RNA-Seq analysis of gene expression of brain endothelial cells has identified members of the Wnt/b-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/b-catenin signaling and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the b-catenin destruction complex or expression of a degradation-resistant b-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17. Conclusions. Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB and they underline the strict reciprocal tuning of this transcription factor and Wnt/b-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.

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Characterization of the blood-brain barrier in genetically diverse laboratory mouse strains

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