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.

Explain how the hypothesis is going to be tested. Aim 1: find out where pericyte and VSMC features co-exist. this should match where contractility ends

Specific Aims

Specific Aim 3: Given the reported absence of aSMA in the higher order capillaries, what proteins could confer pericytes in the middle of the capillary bed contractile ability? Clearing is good because it maintains branch order and enables immunohistochemical labeling. However, there are proteins other than aSMA that enable vessels to constrict...Ira Hermann, D'Amore...peripheral tissues. Other components besides aSMA in contractile machinery. Myosin light chain kinase?? show aSMA in seeDB

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 visualize and excite ChR2-YFP expressed specifically in vascular mural cells.

We 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 in cells that express Cre (Ai32). Because PDGFRBeta is highly expressed in vascular mural cells, this breeding strategy generates offspring with optically-excitable ion channels in vascular mural cells (hereafter called PDGFRBeta-ChR2 animals). In line with previous work, we have found that two photon imaging at an 800 nm laser wavelength produces marked constriction of VSMCs expressing ChR2-YFP, thus indicating that one can simultaneously image vasodynamics and excite ChR2 in a single focal plane. In this aim, we will visualize and excite vascular mural cells through a cranial window, and measure any resultant changes in vessel lumen diameter and velocity as indicated by plasma-labeling fluorescent dye. To categorize where these diameter changes occur along the vascular architecture, 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 vessel diameter and therefore vascular resistance.

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.

Specific aim 1 examines the regions of the vasculature that can alter resting blood flow. It is known that VSMCs rest in a partially-constricted state. During sensory activation of nearby neurons, the VSMCs relax to dilate the vessel and increase local blood flow. It is possible that pericytes are constricted at rest, We predict that pericytes act similarly, and must relax their grip on associated capillaries to permit a sensory-evoked increase in capillary blood flow. We therefore predict that To test this prediction, we will optically depolarizing depolarize pericytes during vibrissae stimulation will prohibit stimulation, effectively prohibiting pericytes from relaxing their grip on the vasculature, thereby preventing hyperemia in associated capillaries.

electric wire analogy - can't let go

expected outcomes and how they relate to hypothesis