Measurement of hydrogen peroxide and oxidant stress in a recirculating whole blood-perfused rat heart model

Aim of StudyIsolated hearts used in the study of ischemia–reperfusion induced myocardial reactive oxygen species (ROS) have typically been perfused with crystalloid buffer. Limitations of crystalloid buffer which may exaggerate the production of ROS, include a requirement for higher oxygen tension and the absence of the intrinsic erythrocyte antioxidant defenses. Using a novel recirculating blood-perfused rat heart model, we measured H2O2 concentration in the blood (as an indicator of ROS formation) and tissue glutathione concentration (an overall measure of oxidant stress) following ischemia and reperfusion.MethodsAutologous blood was obtained and the heart isolated from pentobarbital-anesthetized male Sprague-Dawley rats and placed on a recirculating perfusion circuit with an in-line peristaltic pump and oxygenator. Blood temperature was maintained at 37 °C. Hearts underwent normal perfusion for 120 min (Sham Group, n = 7) or 35 min of normal perfusion, 25 min of global ischemia, followed by 60 min of reperfusion with baseline coronary blood flow levels (IR group, n = 6). Oxygen delivery was compared with a group of buffer-perfused hearts perfused at 85 mm Hg.ResultsLV function in the sham group remained stable for 2 h under normal physiologic oxygen conditions. The oxygen tension and coronary flow were significantly decreased but the myocardial oxygen delivery was significantly increased with blood perfusion compared with buffer perfusion. In the blood IR group, a significant increase in H2O2 was seen early in reperfusion and a reduction in tissue GSH was noted at the end of reperfusion.ConclusionThis model offers significant physiologic advantages in the study of ischemia and reperfusion, particularly in terms of oxygen delivery, compared with the more commonly used acellular buffer-perfused isolated heart systems.