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Characterization of an in vitro model for the study of the short and prolonged effects of myocardial ischaemia and reperfusion in man
journal contributionposted on 2006-07-06, 13:11 authored by Jin-Gang Zhang, Sudip Ghosh, Colin D. Ockleford, Manuel Galiñanes
The mechanisms underlying myocardial ischaemia and reperfusion-induced injury have been investigated, mainly by using animal experimental preparations in vitro and in vivo, but little is known of the process in human myocardium. The present studies characterize an in vitro model using human myocardium for the study of early and delayed effects of ischaemia and reperfusion. The right atrial appendage was manually sliced and incubated in buffer through which was bubbled O2/CO2 (19:1, v/v) for various time periods. Lactate dehydrogenase (LDH) leakage, 3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl-2H-tetrazolium bromide (MTT) reduction, oxygen consumption, nucleotide levels and tissue morphology were all investigated as markers of myocardial injury. The specimens remained stable and viable up to 24 h, but had significantly deteriorated by 48 h. The preparation responded to ischaemia in a time-related manner. Tissue viability was reduced by 25% after 30 min ischaemia, declined to 60% after 60 min ischaemia and to 75% after 120 min ischaemia. Interestingly, the tissue was more susceptible when ischaemia was induced after 24 h of aerobic incubation. The effects of the duration of reperfusion were investigated after a fixed 60 min ischaemic insult. The results of LDH leakage suggest that reperfusion injury is mainly sustained within the first 2 h of reperfusion. However, the results of MTT reduction show that there is a progressive decrease in tissue viability over the 24 h reperfusion period, possibly reflecting the occurrence of tissue necrosis and apoptosis at different reperfusion times. In conclusion, the data provide evidence that the incubation of human atrial tissue in vitro is stable, and slices are viable for at least 24 h, which permits the study of early and delayed consequences of ischaemia and reperfusion in the human myocardium.