MRes Biomedical Research ( in vivo ) ARE ANGIOGENIC STIMULI ADDITIVE ? A STUDY IN THE RAT CORRELATING STRUCTURE AND FUNCTION .

The aim of this study was to elucidate whether two different types of angiogenic stimuli, known to elicit two fundamentally different types of angiogenesis in rat hindlimb skeletal muscle, produce an additive effect when applied simultaneously. When administered in drinking water, the α-1 adrenoreceptor antagonist, prazosin, elevates shear stress causing splitting angiogenesis in extensor digitorum longus (EDL) muscle; surgical extirpation of the tibialis anterior (TA) muscle, a synergist, induces sprouting angiogenesis in EDL muscle due to overload. We used an in vivo rat preparation to investigate how functional readouts of EDL muscle performance and hindlimb blood flow, upon indirect electrical stimulation of the common peroneal nerve, were changed, if at all, once these different types of angiogenesis had been induced. Muscle samples were analysed for structural changes and correlated with function changes, for each treatment group. Prazosin treated animals (2 week administration at 50 mg L) had EDL muscle that had no difference in capillary to fibre ratio (C:F ratio) to that in control animals, contrary to that found in the literature. Neither was there a change in muscle fatigue resistance for an isometric twitch contraction. Extirpated animals (allowed to recover 2 weeks post-operatively) had ipsilateral EDL muscle with an increase in C:F ratio relative to control animals, with a parallel increase in muscle fatigue resistance. Interestingly, combination treated animals (concurrently treated) had ipsilateral EDL muscle with an increase in C:F ratio relative to control animals, although with a lesser increase in C:F ratio than with extirpation alone. The muscle fatigue resistance was not significant when compared to control animals, like it was for extirpated animals. Both extirpated and combination treated animals had contralateral EDL muscles with no difference in C:F ratio or muscle fatigue resistance to that seen in control muscle. Prazosin administered animals (either prazosin treated or combination treated animals) maintained an elevated femoral artery vascular tone to that of non-prazosin administered animals (control and extirpated animals). Irrespective of treatment, no difference in vascular tone profile was discernible during muscle contraction. Thus, the key findings of this study were, that a supramaximal increase in capillary supply by prazosin administration, when there was no metabolic demand for such a change, did not correlate with a functional improvement in muscle fatigue resistance, although our data was confounded by the fact that this treatment did not induce an increase in C:F ratio, like that observed in the literature. Only surgical extirpation led to an increase in muscle fatigue resistance, achieved through a parallel increase in C:F ratio. Interestingly, when both stimuli were applied together, no additive increase in C:F ratio or muscle fatigue resistance was seen; moreover the structural and functional changes were less than that in animals that had undergone extirpation alone. Thus, there seems to be a feedback loop in place that prevents excessive angiogenic stimulation, a putative ‘angiogenic brake’, presumably to prevent signal transduction becoming pathological. When structural and functional data across treatment groups were aligned, it was C:F ratio and not vascular tone that correlated with fatigue index, for an isometric twitch contraction. Thus it seems that for muscle fatigue resistance, local diffusion is more important than global perfusion.