Dynamics of Oxygen Supply Regulation in the Microvasculature

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The microvasculature of every organ of the body is constantly regulating the delivery of oxygen (O2) to every cell in these organs. The goal of this project is to understand how the microvasculature regulates O2 supply such that the capillaries receive enough O2 to supply the nearby tissue. We have evidence that the red blood cell (RBC) which carries O2 to tissue is also responsible for signaling the endothelial cells that form the capillary wall how much O2 each RBC is carrying. The endothelial cells then conduct an electrical signal from the capillaries to the arterioles that control blood flow to either increase or decrease flow depending on whether the O2 levels in the RBCs are changing. If the O2 level in the RBC decreases a signal is conducted upstream to dilate arterioles and increase blood flow. The signaling molecule that the RBCs release is adenosine triphosphate (ATP). This proposal focuses on two questions. Our first goal is to determine how rapidly ATP can be released from the RBC in response to a change in O2 levels.  Our second goal is to determine how rapidly the microvasculature in a living tissue can respond to a change in O2 levels in capillaries. How fast ATP can be released determines how accurately the RBC can report its O2 levels to the capillary. To measure release times we have built "microfluidic" devices (chambers with channels mimicking microvessels) that have small O2 permeable windows which allow us to rapidly change O2 levels of RBCs flowing through the channels. We propose to measure the delay from the change in O2 levels to when ATP is released by detect the light produced when ATP reacts with firefly extract using a very sensitive video camera. How rapidly the microvasculature responds to a change in O2 will give information on all aspects of the regulatory system and confirm whether capillaries can regulate arteriolar blood flow. O2 levels in muscle tissue in rats or mice are changed using a similar microfluidic device in contact with the muscle and the microvascular response is imaged using a microscope and high resolution video cameras. Our third goal is to determine the target volume of muscle tissue for O2 control. The first model of O2 regulation reported 100 years ago and still used today proposed a cylinder of tissue around each capillary as the target volume. Based on our research over the past five years examining the structure of the capillary network around muscle fibres we propose that the target volume is the skeletal muscle fascicle, a bundle of muscle fibres enclosed in a connective tissue layer. The smallest arterioles and venules penetrate the connective tissue and supply a network of capillaries that extend the entire length of the fascicle (and muscle). The knowledge gained from this project will provide new insights into one of the most fundamental regulatory systems in nature and will have far reaching implications in understanding a wide range of cardiovascular diseases.