BONE VIABILITY OF AMPUTATED LIMBS TREATED WITH HYPOTHERMIA:: ASSESSMENT BY EVALUATION OF mRNA LEVELS.

We evaluated rat bone viability using a bone viability index (BVI). To evaluate hypothermic ischaemic bone injury, 21 amputated hind limbs of Fischer rats were preserved at hypothermia (4°C) for 1, 3 and 6 hours. To evaluate hypothermic ischaemia/reperfusion injury, another 28 amputated limbs were transplanted to recipient rats after hypothermic ischaemia for 3 and 6 hours, respectively. Total RNA isolated from each tibia was fractionated by electrophoresis and hybridised with 32P-labelled cDNA of GAPDH, and the radioactivity of intact and degraded GAPDH mRNA measured. BVI was calculated as follows, BVI = {A / (A + B)} × 100, where A and B represent the radioactivities corresponding to intact and degraded GAPDH mRNA bands, respectively. In the hypothermic ischaemic insult group, BVIs were comparable to those of controls. However, in the 3-hour hypothermic ischaemia/reperfusion group, BVI was lower than that of the controls. Likewise, there was a significant difference between the 6-hour ischaemia/reperfusion group and controls. These results showed that bone viability decreased even after just a 3-hour hypothermic ischaemia/reperfusion. [ABSTRACT FROM AUTHOR]