ROUGH DRAFT authorea.com/116265

# Specific Aims

## Goals of the proposed research

Cerebral blood flow impairments are implicated in the pathogenesis of myriad neurological diseases, from multiple sclerosis to dementia. Understanding how cerebral blood flow is regulated under normal conditions may thus permit the development of therapies that can correct blood flow abnormalities and thereby alter the trajectory of neurological diseases. Studies consistently reveal two broad categories of blood flow impairment: 1. abnormal perfusion (hypo- or hyperperfusion), and 2. dampened vasodynamics, defined as a reduced ability of the cerebrovasculature to rapidly change resistance to blood flow in response to environmental shifts such as neural activity, blood pressure, oxygen level, etc. The cells responsible for regulating perfusion and vasodynamics are vascular mural cells, comprised of vascular smooth muscle cells (VSMCs) and pericytes, which adorn the abluminal endothelium. While it is accepted that arterioles ensheathed by vascular smooth muscle cells (VSMCs) are capable of regulating blood flow by modulating vessel diameter, it is debated whether capillaries lined with pericytes can also regulate blood flow. The goal of this dissertation is to clarify the elements of the cerebrovasculature that can regulate blood flow under non-pathological conditions.

## Hypothesis

Pericytes are by definition embedded in the capillary basement membrane, placing them in a perfect position to control capillary blood flow. However, in vivo investigations into the capacity of pericytes to regulate capillary blood flow provide opposing conclusions, placing the field in a stalemate. Hall, et al. (2014) claim that pericytes on capillaries can regulate cerebral blood flow, whereas Hill, et al. (2015) concluded that VSMCs on arterioles can control blood flow, but pericytes on capillaries cannot. Adding to the uncertainty of which cerebrovascular elements regulate blood flow in vivo, these studies defined pericytes and capillaries differently. Hall, et al. (2014) categorized all branches coming from a penetrating arteriole as a capillary, and any vascular mural cell on these branches as a pericyte. Meanwhile, Hill, et al. (2015) identified pericytes and capillaries by the absence of alpha smooth muscle actin (﻿﻿﻿﻿$$\alpha$$SMA), a protein that confers contractile ability to VSMCs. To advance the field beyond this stalemate, we aim to identify which regions of the vasculature (1) are capable of modulating vascular resistance, (2) participate in blood flow changes that occur during sensory-evoked hyperemia, and (3) express contractile proteins such as ﻿﻿﻿﻿$$\alpha$$SMA. Based on preliminary data showing constriction of the vascular lumen in regions of the vasculature that do not express ﻿﻿﻿﻿$$\alpha$$SMA, I hypothesize that capillary pericytes, even those without ﻿﻿﻿$$\alpha$$SMA, can regulate blood flow.

## Specific Aims

Specific Aim 1: To examine the capacity of pericytes and VSMCs to regulate blood flow in isoflurane-anesthetized mice, we will use in vivo two photon microscopy to measure vasodynamics during excitation of ChR2-YFP expressed specifically in vascular mural cells.
To express yellow fluorescent protein-fused channelrhodopsin-2 (ChR2-YFP) in vascular mural cells, we will breed mice expressing Cre recombinase under the control of the promoter for platelet-derived growth factor receptor beta (PDGFRBeta-Cre) with reporter mice that express ChR2-YFP upon Cre recombination. In line with previous work, we have found that two photon imaging of these mice through a cranial window, at an 800 nm laser wavelength, produces marked constriction of VSMCs expressing ChR2-YFP. This indicates that one can use two photon microscopy to simultaneously image vasodynamics and excite ChR2 near the focal plane. In this manner we will image vasodynamics and excite vascular mural cells, and measure resultant changes in vessel lumen diameter and velocity as indicated by plasma-labeling fluorescent dye. To categorize the regions of the vasculature where blood flow modulation occurs under these conditions, we will collect information on branch order for all examined vessels. Branch order is a measure of the number of times a vessel has branched off of the penetrating arteriole. Ultimately, correlating branch order with ChR2-activated diameter and velocity changes will reveal the regions of the vasculature that can modulate blood flow. To control for laser-induced vessel damage that may cause blood flow changes, we will perform identical experiments in animals with YFP, but not ChR2, in the vascular wall. To control for movement artifacts, we will image vasodynamics of all vessels at a wavelength that suboptimally excites ChR2 (900 nm).

Specific Aim 2: To test if pericytes and VSMCs control functional increases in blood flow, we will measure the effect of ChR2 excitation on blood flow velocity changes during vibrissae stimulation in chlorprothixene-anesthetized mice expressing ChR2-YFP in vascular mural cells.
During sensory activation of nearby neurons, VSMCs relax to permit arteriole dilation and a commensurate increase in local blood flow. It is known that sensory stimulation also increases capillary blood flow, but the role of pericytes in capillary hyperemia is debated. We predict that pericytes act similarly to VSMCs, meaning they relax their grip on subjacent capillaries to permit a sensory-evoked increase in capillary blood flow. To test this prediction, we will optically depolarize pericytes while measuring blood flow velocity using line scans during vibrissae stimulation. This will effectively prohibit pericytes from relaxing their grip on the vasculature. If optical depolarization of pericytes prevents hyperemia in associated capillaries, this would suggest that pericytes participate in capillary hyperemia by reducing vascular resistance and permitting an increase in capillary blood flow, similar to the function of VSMCs in arterioles.

Specific Aim 3: To examine if pericytes possess contractile machinery, we will obtain high resolution images of ex vivo brain tissue that has been immunostained and optically cleared.
Studies from our laboratory and others have shown that pericytes four or more branches away from penetrating arterioles do not express ﻿﻿﻿﻿$$\alpha$$SMA. This is inconsistent with our preliminary findings that pericytes at higher branch orders can constrict capillaries and modulate blood flow. Without ﻿﻿﻿﻿$$\alpha$$SMA, how could pericytes in the middle of the capillary bed modulate blood flow? To examine this question, we will use SeeDB, a tissue-clearing technique that enables high-resolution imaging of thick sections of immunostained brain tissue. We will thoroughly investigate which branch orders express ﻿﻿﻿﻿$$\alpha$$SMA and and myosin light chain kinase (MLCK) in vascular mural cells, two essential elements of VSMC contractile machinery.