Characterization of the Skeletal Muscle Microvascular Blood Flow Response to Tissue CO 2 Perturbations using a Gas Exchange Chamber

Abstract

Objective To characterize the skeletal muscle microvascular blood flow response to changing tissue carbon dioxide (CO2) concentrations directly imposed using a microfluidic gas exchange chamber. Methods Eight male Sprague-Dawley rats (145-187g) were anaesthetized using sodium pentobarbital via an intraperitoneal injection. Cannulas were introduced into the left common carotid artery and right jugular vein for blood pressure and heart rate monitoring, administration of maintenance anaesthetic, and fluid resuscitation. The animal was then tracheotomized and mechanically ventilated. The extensor digitorum longus muscle of the right hindlimb was isolated, externalized, and reflected over a microfluidic gas exchange chamber set into the stage of an inverted Olympus IX73 microscope for in vivo visualization of blood flow. The muscle was allowed to equilibrate for 30 minutes before blood sampling and exposure to various gas levels. Using computer-controlled mass flow meters, the muscle was equilibrated with incremental levels of CO2 (0-10%) while maintaining a constant O2 state. One-minute intravital video microscopy sequences of the microcirculation were recorded at a range of CO2 levels (0-10%) while maintaining a constant O2 level. Analysis of hemodynamic variables, red blood cell (RBC) velocity, RBC supply rate (SR), RBC oxygen saturation (SO2), and hematocrit, was conducted offline using custom MATLAB software. Results Both the mean RBC velocity and SR increased incrementally as the CO2 increased. The graded increase of both parameters was significant between the low CO2 levels (0-2%) and moderate-high levels (4-10%); however, there was no significant difference between a physiologically relevant CO2 (5%) and elevated CO2 levels (6-10%) (See Figures 1A & 1B). The graded increase in CO2 had no significant impact on capillary SO2 or hematocrit (See Figures 1C & 1D). Conclusion The preliminary findings of the present study demonstrate the local CO2-sensitivity of the skeletal muscle microvascular system using a novel experimental technique. An expected graded increase in RBC velocity and SR were observed as CO2 increased; however, the lack of significant change following exposure to elevated CO2 suggests a higher relative sensitivity of the microvascular system to changes at low tissue CO2 concentrations. The observed invariance of SO2 at varying CO2 gas concentrations also suggests that local CO2-mediated blood flow regulation is not a result of RBC O2 saturation dependent ATP release. Direct effects of CO2 and the resulting change in pH are known to modulate microvascular blood flow, though the underlying mechanisms of CO2-mediated regulation remain to be defined.