Martou Glyka, O'Blenes Catherine A, Huang Ning, McAllister Sandra E, Neligan Peter C, Ashrafpour Homa, Pang Cho Y, Lipa Joan E
Division of Plastic Surgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
J Appl Physiol (1985). 2006 Nov;101(5):1335-42. doi: 10.1152/japplphysiol.00278.2006.
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.
缺血再灌注（I/R）损伤会导致骨骼肌梗死，而缺血预处理（IPC）可增强动物模型中的缺血耐受性。迄今为止，这在人类骨骼肌中尚未得到证实。本研究旨在建立一种体外模型，以研究模拟IPC对人类骨骼肌的作用效果。将人类骨骼肌条在充氧的Krebs-Henseleit-HEPES缓冲液（37摄氏度）中平衡。分别通过常氧孵育和复氧模拟有氧和再灌注阶段。通过低氧孵育模拟缺血。分别使用ATP、3-（4,5-二甲基噻唑-2-基）-2,5-二苯基-2H-四氮唑溴盐（MTT）和乳酸脱氢酶（LDH）测定评估能量储备、细胞活力和细胞损伤。使用电子显微镜评估形态完整性。研究设计用于测试制剂在24小时常氧孵育下的稳定性（n = 5 - 11）；1、2、3、4或6小时低氧后再复氧2小时的效果；以及在3小时低氧/2小时复氧之前进行低氧预处理（HPC；5分钟低氧/5分钟复氧）的保护作用。在24小时常氧孵育期间，通过MTT、ATP和LDH测定评估，肌条在生理上保持完整。在3小时低氧/2小时复氧后，MTT还原水平降至50.1±5.5%（P < 0.05）。HPC组（82.3±10.8%）和常氧对照组（81.3±10.2%）的MTT还原水平相似，且高于3小时低氧/2小时复氧组（45.2±5.8%）（P < 0.05）。常氧组和HPC组的超微结构形态得以保留，而低氧/复氧组则未保留。这是第一项描述人类骨骼肌稳定体外模型并证明HPC对人类骨骼肌低氧/复氧诱导损伤具有保护作用的研究。
