Viability of ischemia/reperfused muscles in rat: a new evaluation method by RNA degradation.
The rat's skeletal muscle viability was evaluated using the muscle viability index (MVI) which reflects the mRNA degradation. To evaluate ischemic injury of the muscle, 24 hind limbs of Fischer rats (three subgroups of eight rats each) were preserved at normothermia for 1, 3 and 6 h and then tibialis anterior muscle was harvested. To investigate ischemia/reperfusion injury, another 48 limbs were transplanted to recipient Fischer rats after the ischemia at normothermia for 1, 3 and 6 h, respectively. The transplanted muscles were harvested on day 3 and day 7 after transplantation. Eight fresh muscles were also harvested and used as control. Total RNA isolated from each muscle was fractionated by electrophoresis and hybridized with 32P-labelled cDNA of GAPDH, and the radioactivity of intact and degraded GAPDH mRNA was measured. MVI was calculated as follows, MVI = [X/(X + Y)] x 100, where X and Y represent the radioactivities corresponding to intact GAPDH and degraded GAPDH mRNA band, respectively. In 1-h ischemia group, the MVI indices of both ischemic insult and ischemia/reperfusion group were comparable to control. In the 3-h ischemia group, the index of ischemia/reperfused group was comparable to control although the index of ischemic insult group was significantly lower than control. However, in the 6-h ischemia group, both indices of ischemic insult and ischemia/reperfusion group were significantly lower than control. These results show that the muscle damage was detected in ischemia at normothermia even after 3 h. However, this damage was overcome by reperfusion. There was no recovery from damage in muscles that had been preserved for more than 6 h which had resulted in irreversible degeneration. Therefore, in clinical muscle transplantation, one has to transplant the muscle at least within 3-h ischemia.
Akahane
M
M
Department of Orthopedic Surgery, Nara Medical University, Kashihara city, Japan. makahane@naramed-u.ac.jp
Ono
H
H
Ohgushi
H
H
Tamai
S
S
D000818:Animals; D015152:Blotting, Northern; D001831:Body Temperature; D006085:Graft Survival / Q000502:physiology*; D008297:Male; D018482:Muscle, Skeletal / Q000503:physiopathology* / Q000637:transplantation*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D015427:Reperfusion Injury / Q000503:physiopathology*
D012333:RNA, Messenger