Novel technologies and measurement of microvascular blood flow in muscle

In the field of microvascular blood flow, particularly to the skeletal muscle, there has been a considerable history of reference to the incongruity of total limb blood flow and actual myocyte perfusion. Embedded in such an idea is the concept that flow to skeletal muscle should be composed of two physiologically distinct circulations — one that perfuses the myoctes (and therefore may be called "nutritive") and another which must act as a form of physiological shunt or "non-nutritive". The "nutritive and non-nutritive flow" hypothesis was renewed by the muscle research group of the Biochemistry Department at the University of Tasmania to explain perfusion dependent metabolic effects of vasoconstrictors on skeletal muscle. This thesis focuses on the assessment of microvascular perfusion, compared to total flow, in skeletal muscle and in particular in relation to reports of direct and indirect effects of insulin to increase microvascular perfusion of this tissue in vivo. Initially laser Doppler Fluxmetry technology (LDF) was applied to estimate muscle perfusion. For the purposes of verification and comparison two other techniques under development by our group, contrast enhanced ultrasound (CEU) and 1-methyl xanthine metabolism, were also used. In the first section of this thesis the variable of total flow is eliminated by using a constant flow (pump perfused) rat hindlimb. During the application of vasoconstrictors that caused either predominantly nutritive or non-nutritive flow LDF estimation of perfusion was found to be highly variable in spite of the constant total flow. LDF comprises a non vectorial voltage measurement generated by particle movement through a volume of tissue illuminated by the laser light. LDF measurement using a large probe was highly correlated with the metabolic changes induced by changes in nutritive and non-nutritive flow. However, LDF measurement using a smaller implantable probe which measures particle movement in a smaller volume of muscle tissue, showed that there is considerable heterogeneity of response. To better understand the factors that influence changes in the LDF measurement LDF measurements were made of flow controlled through fabricated glass capillary arrays of various architectures. The third study addressed changes induced by insulin in vivo, in anaethetised rats during a hyperinsulinaemic euglycaemic clamp. Using a large surface LDF probe the LDF signal from the muscle increased during the administration of insulin. In contrast the LDF signal did not increase during epinephrine administration even though the epinephrine caused a comparable increase in femoral artery blood flow as seen with insulin. Furthermore the time course of LDF change in the presence of insulin was more closely aligned with changes in glucose uptake than it was with changes in femoral blood flow. In the final series of experiments using LDF, changes due to insulin in human muscle was assessed. The results suggested that physiological hyperinsulinaemia stimulated not only total blood flow and skin microvascular perfusion, but also augmented human skeletal muscle microvascular recruitment and vasomotion as detected directly by laser Doppler measurement. In the second technique, CEU, microbubbles (an albumin membrane encapsulating an inert gas in the form of a 4 micron diameter sphere) act as contrast when tissue is visualised using ultrasonography. Analysis of the rate and extent of contrast (microbubble) appearance during imaging of the target tissue allows us to calculate indicators of the microvascular velocity and microvascular volume. The third technique based on capillary endothelial metabolism of the exogenous substrate 1-methylxanthine, was developed at the University of Tasmania. 1-Methylxanthine is a reporter substrate, which as it passes through the nutritive capillary route of the muscle is metabolized to 1-methyl urate; metabolism is proportional to available capillary surface area. To consolidate the LDF findings, the two additional techniques for assessing changes in capillary recruitment were used. Once again, the hyperinsulinaemic euglycaemic clamp was used but on this occasion a physiologic dose of insulin was infused. Microvascular recruitment (measured by 1-MX metabolism as well as by CEU) increased during insulin administration. Furthermore, at certain time points microvascular recruitment occurred without changes to femoral artery flow. From these studies it was evident that insulin increases tissue perfusion by recruiting microvascular beds, and at physiological concentrations this precedes increases in total muscle blood flow by 60-90 min. Since there was no change in the mean red cell velocity, it seem likely that insulin had increased capillary recruitment by redistributing blood flow from other vessels possibly non-nutritive. In the final study capillary recruitment was again measured by the metabolism of infused 1-methyl xanthine and used to assess responses to insulin in normal colony rats as well as lean and obese Zucker rats (an animal model for type 2 diabetes). It was concluded that muscle insulin resistance of obese Zucker rats is accompanied by impaired hemodynamic responses to insulin including markedly impaired capillary recruitment and femoral blood flow when compared to lean Zuckers or colony control rats. Taken together, the findings embodied by this thesis show that the three techniques of LDF, CEU and 1-MX metabolism, each detect a vascular effect of insulin in vivo, characterized by capillary recruitment. This was detectable at physiologic insulin and occurred in both muscle of rats and humans. In addition, LDF added a further aspect of insulin action of an insulin-mediated increase in vasomotion at low frequency, very likely suggesting increased neural activity.