Intravital Video Microscopy of Forelimb Capillary Blood Flow in Rat Skeletal Muscle

Abstract

ObjectiveThe present study describes the development of a novel in vivo microvascular preparation for the investigation of capillary blood flow in the extensor carpi radialis longus (ECRL) muscle of rat. Blood flow measurements in the forelimb provide an alternative to other contemporary vascular preparations and is particularly useful for investigating capillary blood flow in disease models where hind limb flow is impaired (e.g. hind limb unloading, reduced uterine perfusion pressure etc.).MethodsTwo male Sprague‐Dawley rats, 174 and 161g were anaesthetized with pentobarbital, tracheotomized, ventilated, and maintained in a normotensive state via minimal saline infusion in the jugular vein. The skin on the lateral side of the right forelimb was removed exposing the muscle capsule surrounding the extensor carpi radialis longus and brevis muscles. The distal tendon of the ECRL was secured with a silk ligature and the muscle was carefully isolated and reflected onto an inverted microscope stage. The muscle was bathed in warm saline and isolated from the air via polyvinylidene chloride film and a glass coverslip. Ten – twenty, 1 minute video sequences of capillary blood flow at 20X magnification were recorded in each animal. Videos were analyzed offline using custom software written in MATLAB yielding frame‐by‐frame measurements of capillary hemodynamics and oxygen saturation.ResultsCapillary measurements were made in a total of 69 vessels (31 ‐ 38 in each animal), with erythrocyte velocities of 196.8±40.2 μm/s; tube hematocrit 19.9±2.9 %; 10.7±0.7 cells/s; and entrance and exit oxygen saturation of 70.4±5.6 % and 55.8±4.6% respectively.ConclusionsWe demonstrate direct measurement of microvascular hemodynamics and capillary oxygen saturation in a novel intravital preparation using rat ECRL muscle.