Measuring blood flow in the skeletal muscle microcirculation using laser speckle flowmetry

The presence of a native collateral circulation, which serves as a natural bypass for blood flow around an occlusion, improves prognosis for patients with ischemic diseases, such as peripheral arterial occlusive disease (PAOD). However, not all patients have a native collateral circulation, and animal models suggest a genetic basis for this variability. In mice, such as the BALB/c, that lack native arteriolar collaterals, neocollateral formation from capillaries that connect two arterial trees can occur after arterial occlusion, resulting in reperfusion of the ischemic watershed. Immature arterialized collateral capillaries (ACCs) at 7 days post arterial occlusion do not vasodilate in response to physiological stimuli and are therefore unable to match blood flow with metabolic demand, but mature ACCs at 21 days exhibit normal vascular reactivity. Therefore we wanted to determine if vasodilation of ACCs at 21 days post arterial occlusion is capable of increasing flow throughout the ischemic arteriolar tree, because the ACCs are small-caliber vessels feeding progressively larger arterioles. One aspect of blood flow, vessel diameter, is measured routinely in our lab using bright field intravital microscopy; however blood velocity is more challenging to measure in the spinotrapezius microvasculature. In particular, we wanted to assess vasculature-wide changes in blood flow, which cannot be accomplished using laser Doppler flowmetry due to its small field of view or particle image velocimetry due to the curvature of the spinotrapezius. Therefore, we adapted a laser speckle flowmetry (LSF) protocol to measure blood velocity in the spinotrapezius microvasculature. In LSF, the scattering of laser light incident on the muscle produces a characteristic speckle. This speckle changes over time as erythrocytes flow through the vasculature of the muscle. If captured over the finite exposure time of a detector, the speckle is blurred, and the degree of blurring is related to the speed at which the erythrocytes are flowing through the vasculature. LSF yields velocity information across the entire field of view, and multiple fields of view can be stitched together to create a velocity map of the spinotrapezius vasculature. Using LSF, in conjunction with bright field intravital microscopy, we measured blood velocity and blood vessel diameter in vivo to quantify changes in blood flow. We found that vasodilation of mature ACCs (i.e. at day-21) increases blood flow (288 ± 72%) in the ischemic tree, which is comparable to the contralateral, control arterial tree (168 ± 76%), while vasodilation of immature ACCs (i.e. at day-7) does not increase blood flow (17 ± 27%) in the ischemic tree. The ability of mature ACCs to increase flow in the ischemic tree supports their potential as a therapeutic target for patients with PAOD who lack native collateral vessels. Future work will include the use of a contrast agent to provide more detailed vessel analysis (e.g. branch order effects), and similar analyses on mice with relevant comorbidities such as diabetes mellitus and hypercholesterolemia to study any potential impairment in arterialization and outward remodeling