Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries.

Introduction: The vasomotor response of the coronary artery is altered by hypoxia-reoxygenation (H-R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H-R injury.

Methods: Porcine coronary arterial rings were isolated and placed in Krebs-Henseleit (K-H) solution. Rings were exposed to normoxic conditions (control group) and two different H-R conditions: the first induced by a 95% N(2)-5% CO(2) gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 degrees C) hypoxia-reoxygenation in an air-tight beaker filled with K-H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K-H solution aerated with a 95% O(2)-5% CO(2) mixture under normothermic (37 degrees C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferroni's post-test.

Results: Distal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced (p<0.001) from the values for normoxic rings (81.0+/-1.0%, n=30) after 40-min H-R (50.5+/-5.3%, n=30), 60-min H-R (32.1+/-3.5%, n=30), 24-hours hypothermic H-R (56.0+/-2.3%, n=30) and after 48-hours hypothermic H-R (38.5+/-5.1%, n=30).

Conclusions: The model employing 40-min normothermic H-R is as effective as 24-hours hypothermic H-R, and 60-min normothermic H-R as 48-hours hypothermic H-R for studying the deleterious effects of H-R on EDHF-mediated relaxation.