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Local Mild Hypothermia (30-32°C) Is Effective In Protection Of Ex Vivo Human Skeletal Muscle From Hypoxia/reoxygenation Injury
Anne O’Neil, M.D., Ph.D., F.R.C.S.(C).1, Stefan Hofer, M.D., Ph.D., F.R.C.S.(C).2, Homa Ashrafpour, B.Sc.1, Ning Huang, M.D.1, Toni Zhong, M.D., F.R.C.S.(C).2, Christopher Forrest, M.D., M.Sc., F.R.C.S.(C).1, Cho Y. pang, Ph.D.1.
1Hospital for Sick Children, Toronto, ON, Canada, 2Toronto General Hospital, Toronto, ON, Canada.
Introduction: In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion which may lead to significant ischemia/reperfusion injury. Other investigators reported that low local hypothermia (local cooling at 4-10°C) significantly reduced ischemia/reperfusion injury in skeletal muscle of different species of laboratory animals. However, this range of severe low local hypothermia is known to induce capillary damage. More recently, other investigators reported that low local mild hypothermia at 32-34°C significantly reduced ischemia/reperfusion injury in rabbit rectus femoris muscle in vivo. However, this infarct protective effect of low local hypothermia has not been tested in human skeletal muscle. The objective of this study was to use our established ex vivo human skeletal muscle culture model to study the efficacy of low local mild hypothermia (30-32°C) in salvage of human skeletal muscle from hypoxia (ischemia)/ reoxygenation (reperfusion) injury.
Methods: Human rectus abdominus muscle strips ( ̴ 0.5 X 0.5 X 15mm) derived from muscle biopsies were cultured in Krebs buffer bubbled with 95% N₂ / 5% CO₂ (hypoxia) or 95% O₂/5% CO₂ (reoxygenation). Control muscle strips underwent 6h normoxia at 37°C (normal thermia). Other skeletal muscle groups underwent 4h hypoxia/2h reoxygenation in normal thermia at 37°C or in low local hypothermia at 32°C, 30°C or 10°C. Skeletal muscle viability, injury, and energy content were assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and adenosine triphosphate (ATP) content, respectively.
Results: The MTT reduction in the skeletal muscle of the normoxic control group was 0.66 ± 0.08 optical density (OD)/mg wet wt. This MTT reduction was significantly reduced to 0.24 ± 0.03 OD/mg wet wt. (n=6; p<0.05) in the treatment group subjected to 4h hypoxia/2h reoxygenation at 37°C. Importantly, this MTT content was significantly (p<0.05) increased in the treatment groups (n=6) subjected to 4h hypoxia/2h reoxygenation at 32°C (0.42 ± 0.04 OD/mg wet wt.; n=6), at 30°C (0.44 ± 0.42 OD/mg wet wt.; n=6) and at 10°C (0.39 ± 0.05 OD/mg wet wt.; n=6). There was no significant difference in MTT content among these three hypothermial treatment groups. The skeletal muscle contents of LDH and ATP are being analyzed at the present time, and will be ready for presentation at the meeting.
Conclusion: The data which we have generated thus far indicate for the first time that local mild hypothermia between 30°C to 32°C is effective in salvage of ex vivo human skeletal muscle from hypoxic/reoxygenation injury compared with the severe 10°C hypothermia. The mechanism of local mild hypothermia (30°C to 32°C) is being studied in our laboratory. Our results support the design of clinical study of local mild hypothermia (30-32°C) for prevention and/ or salvage of skeletal muscle from ischemia/ reperfusion injury in transplantation or replantation surgery.
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