Advanced in vitro approach to study neurovascular coupling mechanisms in the brain microcirculation

Key points • We show that parenchymal arterioles of less than 30 μm in diameter can be successfully cannulated and perfused in cortical and hypothalamic brain slices. • Increased flow/pressure induced parenchymal arteriolar constriction. • Alternatively to video microscopy, diameter measurements of cannulated arterioles could be obtained from perfusion velocity values obtained using fluorescence recovery after photobleaching. • Perfused and pressurized parenchymal arterioles responded with dilatation to two well‐established signals in neurovascular coupling, K+ (10 mM) and activation of metabotropic glutamate receptors (mGluRs). An understanding of the signalling events underlying neurovascular coupling mechanisms in the brain is a crucial step in the development of novel therapeutic approaches for the treatment of cerebrovascular‐associated disorders. In this study we present an enhanced in vitro brain slice preparation from male Wistar rat cortical slices that incorporates haemodynamic variables (flow and pressure) into parenchymal arterioles resulting in the development of myogenic tone (28% from maximum dilatation). Moreover, we characterized flow‐induced vascular responses, resulting in various degrees of vasoconstrictions and the response to 10 mm K+ or astrocytic activation with the mGluR agonist, t‐ACPD (100 μm), resulting in vasodilatations of 33.6 ± 4.7% and 38.6 ± 4.6%, respectively. Using fluorescence recovery, we determined perfusate velocity to calculate diameter changes under different experimental pH conditions. Using this approach, we demonstrate no significant differences between diameter changes measured using video microscopy or predicted from the velocity values obtained using fluorescence recovery after photobleaching. The model is further validated by demonstrating our ability to cannulate arterioles in two brain regions (cortex and supraoptic nucleus of the hypothalamus). Altogether, we believe this is the first study demonstrating successful cannulation and perfusion of parenchymal arterioles while monitoring/estimating luminal diameter and pressure under conditions where flow rates are controlled.