Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.
Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.
Martou
Glyka
G
Division of Plastic Surgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
O'Blenes
Catherine A
CA
Huang
Ning
N
McAllister
Sandra E
SE
Neligan
Peter C
PC
Ashrafpour
Homa
H
Pang
Cho Y
CY
Lipa
Joan E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival; D005562:Formazans / Q000378:metabolism; D006801:Humans; D019194:Ischemic Preconditioning; D032342:Muscle Cells / Q000378:metabolism / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D009924:Organ Culture Techniques / Q000379:methods; D010100:Oxygen / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology*; D013778:Tetrazolium Salts / Q000378:metabolism
D005562:Formazans; D013778:Tetrazolium Salts; C079782:MTT formazan; D000255:Adenosine Triphosphate; D010100:Oxygen