Pericyte contractility, microvascular blood flow and the consequences of stroke
Creators: name: Brown, Lachlan
Publisher: name: University of Tasmania
Year: 2024

description: <b>Scope and Aims</b>Several common diseases, including ischemic stroke, display pathological changes in cerebral blood flow. Recent advances in imaging have revealed that blood flow regulation in the brain begins at the capillary level, not the arteriole level. A specific type of cell known as a pericyte has been found responsible for control of blood flow at the capillary. Pericytes reside on the vessel wall of capillaries and promote the communication between the vasculature and the brain parenchyma. Because of their location and role, pericytes provide a promising target to treat pathological changes in cerebral blood flow. Due to the recency of these findings, pericyte and vascular physiology underlying blood flow in both healthy and diseased states including ischemic stroke is yet to be fully explored. This is partly due to a lack of appropriate models to study pericyte contractility and physiology, and a sparsity of research on pericyte and vascular change after ischemic stroke.<br>Therefore, I aimed to characterise pericytes in an in vitro cell culture setting and develop a live imaging single cell pericyte contractility assay to identify contractile and relaxant mechanisms of pericytes. Next, I aimed to develop and optimise a methodology to detect and analyse blood vessels, pericytes and their interactions in the mouse brain, and to determine whether there were changes in pericytes and vessels across a number of brain regions. Next, I aimed of to explore how pericytes contract under ischemic conditions <i>in vitro</i>, and how they modulate the microvasculature and blood flow after ischemic stroke. Finally, I aimed to determine whether pharmaceutical intervention with a mammalian target of rapamycin (mTOR) inhibitor could prevent these pericyte and vessel changes.<b>Methodology</b>Human brain vascular pericytes (HBVP) were cultured and underwent immunocytochemistry, morphological analysis, and single cell live imaging. Contractility was measured by change in pericyte membrane area using differential interference contrast microscopy, and cytosolic Ca<sup>2+</sup> levels were quantified using fluorescent ratiometric Ca<sup>2+ </sup>imaging. To detect pericytes and vessels <i>in vivo</i>, a NG2-DsRed mouse line was utilised alongside vascular labelling with FITC-albumin gel casting and several methods of vascular labelling, including transcardial and tissue slice application of isolectin. Ischemia was chemically modelled <i>in vitro</i>, while ischemic stroke was modelled <i>in vivo</i> in NG2-DsRed mice using middle cerebral artery occlusion (MCAO). Cerebral blood flow was measured during MCAO using laser Doppler (LD) flowmetry. Quantification of pericyte and vessels <i>in vivo</i> was achieved through development of novel semi-automated image analysis tools in open-source image analysis programmes ImageJ and QuPath.<b>Results</b>Cultured HBVP expressed known proteins that are considered markers of pericytes and that multiple pericyte morphologies existed in culture. These morphologies changed as passage number increased, and pericytes with a ‘standard’ morphology displayed characteristics and expressed proteins that are most associated with contractile vascular pericytes. I generated a working contraction-relaxation model for pericytes i<i>n vitro</i> and discovered differential effects between vasoactive agents including noradrenaline and endothelin-1 in their ability to contract pericytes and their relationship with intracellular calcium changes. In addition, I identified that blocking mTOR with rapamycin could prevent NA-mediated pericyte contraction, a novel mechanism that is targetable. Following this, NG2-DsRed mice were used to visualise pericytes <i>in vivo</i>, while FITC-albumin gel casting and isolectin B4 labelling provided measures of total perfused vasculature and total vasculature. Pericyte number and their coverage of blood vessels as well as a number of vascular parameters (including length, area, and width) were not shown to differ between multiple regions of the brain. In the context of ischaemia, pericytes were shown to contract during chemical ischaemia <i>in vitro</i>, which was blocked in the presence of rapamycin, further confirming mTOR as a target for pericyte contractility. <i>In vivo</i> experiments using the MCAO model of ischemic stroke showed reduced vascular perfusion and pericyte number and signal in the ischemic core relative to the contralateral hemisphere and sham animals at 30 mins of reperfusion following 60 mins ischaemia. Rapamycin treatment showed a slight increase in blood flow after reperfusion but no changes in the microvasculature or pericytes were observed.<b>Major conclusions</b>My research has advanced our understanding of pericyte contractility and vascular changes acutely following stroke, and has provided several tools that will be able to further enhance our understanding of pericyte biology and vessel interactions important for brain function. Firstly, my single-cell contractility assay provides a novel and direct measure of pericyte cell area that can be used to better understand mechanisms involved in pericyte contractility and relaxation. Secondly, my characterisation of pericyte subtypes<i> in vitro</i> adds to the evidence that pericyte subtypes are functionally important to contraction, while my<i> in vivo</i> data showed that vascular location can be used to further define the pericyte subtypes. Thirdly, I have reported pericyte and vascular changes at the hyperacute timepoint after stroke and reperfusion, which has important ramifications for the ongoing development of tissue injury and functional disability following stroke., Therefore, understanding of the acute vascular pathology post-stroke is vital for the field. Finally, I have shown that rapamycin inhibits pericyte contraction in response to the vasoconstrictor noradrenaline as well as ischaemia <i>in vitro</i>. While treatment with rapamycin after stroke <i>in vivo</i> did not reveal major vascular effects, further analysis of brain regions and timepoints may reveal critical vasculoprotective effects mediated by rapamycin. This research is an important step in the generation of novel tools and knowledge needed to improve our understanding of pericytes and the microvasculature in the brain, and to develop novel treatment strategies for ischemic stroke and other vascular-associated diseases.<br>