Isolation of Rodent Brain Vessels

Background

The brain is highly dependent on a constant supply of oxygen and nutrients that arrive through a vast network of blood vessels. The blood-brain barrier (BBB), mainly composed of microvascular endothelial cells that line cerebral microvessels along with periendothelial structures, which include pericytes, astrocytes and a basement membrane (Saraiva et al., 2016; Librizzi et al., 2017), guarantees the control of an homeostatic environment, necessary to maintain the health of brain cells. Thus, the study of how certain pathologies that can interfere with the integrity of cerebrovasculature is of great importance. Indeed, strong evidence from clinical, imaging, epidemiological and neuropathological studies confirmed over the past two decades that the presence of cerebrovascular disease has a pivotal role in Alzheimer disease (AD) and other dementias associated with aging (Chui et al., 2006; Schneider et al., 2007; Gorelick et al., 2011; Wharton et al., 2011; Yarchoan et al., 2012; Bennett et al., 2013; DeCarli, 2013; Toledo et al., 2013; Yates et al., 2014). Besides the low number of papers dedicated to the study of isolated brain vessels, the isolation protocols used in those studies present some inconsistencies rendering difficult the comparison and interpretation of the published observations. With this protocol, we intend to offer a standardized procedure to help researchers working in this field. This protocol was adapted from a previous protocol described by McNeill et al. (1999) and used in our laboratory to isolate total (arterial and venous) brain vessels from rodents (Figures 1 and 4) (Carvalho et al., 2010; Carvalho et al., 2013; Plácido et al., 2017).


Figure 1. Evaluation of the activity of the mitochondrial enzymatic complexes of mice brain vessels. Mitochondrial complexes I-III (A), II-III (B) and IV (C) were determined in vessels isolated from the brains of 11-month-old male wild type (WT; C57BL6/129S), type 2 diabetes-like mice (WT mice exposed to 20% sucrose solution during 7 months) and triple transgenic mice for Alzheimer disease (3xTg-AD, B6;129-Psen1 Tg(APPSwe,tauP301L)1Lfa/Mmjax). A significant decrease in the activity of mitochondrial complexes I-III was observed in brain vessels isolated from 3xTg-AD and type 2 diabetes-like mice. Also, a significant decrease in the activity of complex IV was observed in brain vessels isolated from 3xTg-AD mice. Data shown represent mean ± SEM from 6-8 pools of n = 3. Statistical significance: *P < 0.05; **P < 0.01 when compared with WT mice. Statistical significance was determined using the paired Student’s t-test and Kruskal-Wallis test for multiple comparisons, followed by the posthoc Dunn test (GraphPad Prism 5). These graphs have been previously published in Journal of Alzheimer’s Disease (DOI: 10.3233/JAD-130005) with permission from IOS Press.