Quantitation of postischemic skeletal muscle necrosis: Histochemical and radioisotope techniques

Abstract

Skeletal muscle necrosis will result from prolonged periods of ischemia. The purpose of this study was to develop a method to estimate the extent of necrosis using nitroblue tetrazolium staining and technetium scanning. The bilateral canine gracilis muscle preparation with total vascular isolation was exposed to 4 hr of complete normothermic ischemia followed by reperfusion. After 45 hr of reperfusion 99mTc pyrophosphate (PYP) was injected and 3 hr later the muscles were harvested, cut into six slices, and stained with nitroblue tetrazolium. Biopsies were taken from tetrazolium-positive and -negative areas for electron microscopy to confirm the ability of the stain to distinguish viable from necrotic muscle. Computerized planimetry of the staining pattern was used to estimate the extent of necrosis as a percentage of the total muscle. Electron microscopy confirmed the validity of nitroblue tetrazolium to discriminate between viable and necrotic skeletal muscle in this experimental model. After 4 hr of ischemia the percentage necrosis was 30.2 ± 6.1% (mean ± SEM, n = 12), there was no difference in the extent of necrosis in left vs right paired muscles, using tetrazolium staining or technetium PYP uptake. There was a statistically significant correlation between the percentage necrosis and the density of 99mTc PYP uptake per muscle ( r = 0.83, P < 0.001) and per slice ( r = 0.94, P < 0.001). This study demonstrates the ability of tetrazolium staining to accurately differentiate between viable and necrotic skeletal muscle and provides a reproducible method for estimating the extent of necrosis in the gracilis muscle model.