Analysis of ischemic stroke-mediated effects on blood–brain barrier properties along the arteriovenous axis assessed by intravital two-photon imaging

Abstract

Early breach of the blood–brain barrier (BBB) and consequently extravasation of blood-borne substances into the brain parenchyma is a common hallmark of ischemic stroke. Although BBB breakdown is associated with an increased risk of cerebral hemorrhage and poor clinical prognosis, the cause and mechanism of this process are largely unknown. The aim of this study was to establish an imaging and analysis protocol which enables investigation of the dynamics of BBB breach in relation to hemodynamic properties along the arteriovenous axis. Using longitudinal intravital two-photon imaging following photothrombotic induction of ischemic stroke through a cranial window, we were able to study the response of the cerebral vasculature to ischemia, from the early critical hours to the days/weeks after the infarct. We demonstrate that disruption of the BBB and hemodynamic parameters, including perturbed blood flow, can be studied at single-vessel resolution in the three-dimensional space as early as 30 min after vessel occlusion. Further, we show that this protocol permits longitudinal studies on the response of individual blood vessels to ischemia over time, thus enabling detection of (maladaptive) vascular remodeling such as intussusception, angiogenic sprouting and entanglement of vessel networks. Taken together, this in vivo two-photon imaging and analysis protocol will be useful in future studies investigating the molecular and cellular mechanisms, and the spatial contribution, of BBB breach to disease progression which might ultimately aid the development of new and more precise treatment strategies for ischemic stroke.