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

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. Overall design: 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 RNA sequencing of brain endothelial cells.

群体中的遗传变异对单基因疾病及多因子疾病的表型表达均存在调控作用，其潜在的遗传贡献依赖于多个相互作用的变异位点。当前用于人类疾病建模的常见实验室小鼠品系，缺乏人类群体所具备的遗传多样性，因此啮齿类动物研究结果与人类疾病的相关性较为有限。
脑血管的功能状态是脑部疾病的重要修饰因子。尤为关键的是，血液与脑组织之间的限制性界面——血脑屏障（blood-brain barrier, BBB）是中枢神经系统（Central Nervous System, CNS）药物递送的主要障碍。
本研究采用遗传多样性各异的小鼠品系，旨在解析不同近交系小鼠完整血脑屏障的表型与转录组变异特征。
我们通过多维度表型分析手段，对近期野生来源小鼠品系（CAST/EiJ、WSB/EiJ、PWK/PhJ）以及长期近交小鼠品系（129S1/SvImJ、A/J、C57BL/6J、DBA/2J、NOD/ShiLtJ）的脑血管异质性进行了研究。具体实验方法包括：借助免疫组织化学（immunohistochemistry）与共聚焦激光显微镜（confocal laser microscopy）结合定量图像分析，检测大脑皮层与海马体两个脑区的血管密度及周细胞（pericyte）覆盖率；采用低分子量荧光示踪剂荧光素钠（sodium fluorescein）结合分光光度分析，评估选定品系年轻与老年小鼠的血脑屏障通透性。
为进一步表征近交系小鼠脑内皮细胞的表型特征，我们对从大脑皮层与海马体中分离并分选得到的内皮细胞进行了批量RNA测序（bulk RNA sequencing）。
研究结果显示：所有受试小鼠品系的大脑皮层与海马体血管密度及周细胞覆盖率均无显著差异；不同品系小鼠的内皮细胞动静脉分区标记物染色模式较为相似；不同品系小鼠对小分子荧光示踪剂荧光素钠的血脑屏障通透性亦无明显差异。内皮细胞转录组学分析结果表明，动物性别是导致基因表达差异的主要影响因素；此外，与内皮细胞功能及血脑屏障生物学相关的多个基因的表达水平，在野生来源与长期近交小鼠品系间存在显著差异。
本研究分析结果表明，尽管受试小鼠品系及不同性别间的脑实质血管形态与血脑屏障通透性均无显著差异，但脑内皮细胞的转录组差异提示完整血脑屏障的基因表达存在变异。这些基础水平的表达差异可能在病理状态下成为混杂因素，进而导致基于性别或遗传多态性的不同功能结局。
实验整体设计：本研究通过对脑内皮细胞进行RNA测序，探究了近期野生来源小鼠品系（CAST/EiJ、WSB/EiJ、PWK/PhJ）与长期近交小鼠品系（129S1/SvImJ、A/J、C57BL/6J、DBA/2J、NOD/ShiLtJ）的脑血管异质性。