A model for determining baseline morphometrics of the capillary bed in skeletal muscle

Changes in capillary diameter (CD) have been used to detect the effect of pharmacological interventions, physical treatments and ischaemic reperfusion injury in skin and muscle. For comparison, CDs within similar structures in the untreated limb of the same subject have been used as the control, with the assumption that CDs are the same in both limbs. This study employs light microscopic morphometry (LMM) to explore this premise, investigates the repeatability of LMM and determines the number of CDs that should be measured to produce a reliable result. Muscle biopsies were obtained from the left (L) and right (R) vastus lateralis of four rabbits and the left and right tibialis anterior of three vervet monkeys. Thin (1-micro m) resin sections were prepared for LMM examination and CDs were measured using computerised image analysis. To determine the repeatability of measurement, two specimens were re-measured on five occasions at monthly intervals. The mean CD of each biopsy from L and R limbs were compared and the number of measurements necessary to produce a result with less than a 3% difference was determined. A minimum of 58 and a maximum of 175 CDs were measured from each specimen. Repeated measurement of the same biopsies showed a non-significant mean difference of less than 2% in CD means between the first measurement and each of the four subsequent measurements. There was a non-significant difference of 3.7% in CD means between R and L limbs in one animal, in which less than 70 CDs were measured on each side. When 100 CDs or more were measured, the difference was always less than 2.3%. When grouped, the difference in CD means between L and R limbs was 0.5% in rabbits and 2.0% in vervet monkeys. The results confirm that firstly, CD data derived from a muscle in an untreated limb can be used as a control for experiment-mediated changes of CD in the other; secondly, LMM appears to be a reliable means of measuring CD; and thirdly, while fewer measurements may suffice, ideally 75-100 CDs are needed to provide a dependable result.