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COMBINED HYPOXIC PRECONDITIONING AND POSTCONDITIONING DO NOT CONFER ADDITIVE PROTECTION OF EX VIVO HUMAN SKELETAL MUSCLE FROM ISCHEMIA/REPERFUSION INJURY
Presenter: Presenter: Andreas Naparus, MRCS MBBS BSc (Hons) AICSM
Co-Authors: Ashrafpour H; Huang N; Hofer SO; Zhong T; Forrest CR; Pang CY
The Hospital for Sick Children Toronto

Introduction: Prolonged ischemia frequently occurs during reconstructive surgery, causing irreversible ischemia/reperfusion (I/R) injury, even when successful revascularisation is achieved. We previously reported that brief cycles of hypoxia/reoxygenation before or after sustained hypoxia (hypoxic pre- and postconditioning, HPreC and HPostC, respectively) effectively protected ex vivo human skeletal muscle from I/R injury. In elective surgery, both HPreC and HPostC are possible. At present, animal studies are inconclusive which is better or whether there is an additive effect when combined. Moreover, no human tissue studies have been reported.

Methods: Human rectus abdominis muscle strips were cultured in Krebs buffer bubbled with 95%N2/5%CO2 (hypoxia) or 95%O2/5%CO2 (normoxia). Controls underwent 5h normoxia. Other groups underwent 3h hypoxia/2h reoxygenation and were treated with HPreC, HPostC (induced by 1 cycle of 5min hypoxia/reoxygenation before or after hypoxia, respectively), HPreC+HPostC or no treatment. In addition, atractyloside, a mitochondrial permeability transition pore (mPTP) opener was added to other treatment groups. Muscle injury, viability, and ATP synthesis were assessed by LDH release, MTT reduction and ATP content, respectively.

Results: LDH release increased and MTT reduction and ATP content decreased after 3h hypoxia/2h reoxygenation (p<0.05; n=7 patients) compared to normoxic controls. HPreC, HPostC or combined HPrec+HPostC were similar to normoxic controls and each other. Atractyloside completely abolished the protective effect but did not significantly alter untreated groups.

Conclusion: We conclude that HPreC and HPostC are equally effective in protection of ex vivo human skeletal muscle against I/R injury. Morevoer, there is no additive effect in combination because they both act by closing the mPTP. The potent effect of HPostC alone suggests a clinical application of ischemic postconditioning for salvage of human tissue from reperfusion injury in transplantation and replantation surgery, obviating the need for prophylaxis and dramatically reducing numbers needed to treat.


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