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Viability of ischemia/reperfused muscles in rat: a new evaluation method by RNA degradation
1Department of Orthopedic Surgery, Nara Medical University, Kashihara city, Japan. makahane@naramed-u.ac.jp
Abstract:
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.
Insights
Skeletal muscle viability decreases with ischemia, but reperfusion can overcome damage up to 3 hours. Beyond 6 hours, muscle degeneration is irreversible, necessitating timely transplantation.
Area of Science:
- Biomedical Engineering
- Surgical Research
- Tissue Engineering
Background:
- Skeletal muscle viability is critical for successful transplantation.
- Assessing muscle damage after ischemia and reperfusion is essential for clinical practice.
Purpose of the Study:
- To evaluate skeletal muscle viability after normothermic ischemia and ischemia/reperfusion.
- To determine the critical time limit for muscle transplantation to prevent irreversible damage.
Main Methods:
- The muscle viability index (MVI), based on GAPDH mRNA degradation, was used to assess muscle damage.
- Fischer rats underwent hind limb ischemia (1, 3, 6 hours) with or without transplantation to assess ischemia/reperfusion injury.
- Muscle samples were analyzed for intact and degraded GAPDH mRNA radioactivity.
Main Results:
- Muscle viability was maintained after 1-hour ischemia but significantly decreased after 3-hour ischemia without reperfusion.
- Reperfusion successfully restored viability after 3-hour ischemia, but not after 6-hour ischemia.
- Significant muscle damage and irreversible degeneration were observed after 6-hour ischemia, even with reperfusion.
Conclusions:
- Normothermic ischemia causes detectable muscle damage within 3 hours.
- Reperfusion can mitigate ischemia-induced muscle damage if performed within 3 hours.
- For successful clinical muscle transplantation, ischemia time should be limited to within 3 hours to avoid irreversible degeneration.