Failure of perfusion with oxygenated Krebs‐Ringer solution to preserve the eccrine function of the vascular endothelium in bone

An ex vivo canine tibia preparation was perfused at a constant rate with aerated (95% O2-5% CO2) Krebs-Ringer solution for 24 h. Bolus injections of norepinephrine (0.125-0.5 micrograms) were given and then acetylcholine (5 x 10(-5) M) was used to stimulate endothelial production of smooth muscle relaxing factors. Following 1 h of perfusion the addition of acetylcholine resulted in significant attenuation of the response to norepinephrine (p < 0.001). After 4 h perfusion acetylcholine did not attenuate the norepinephrine response, but addition of L-arginine (the precursor of endothelial-derived relaxing factor) resulted in significant attenuation in the presence of acetylcholine (p < 0.005). At 6, 12, and 24 h the acetylcholine did not attenuate the norepinephrine response. It is concluded that normothermic, continuous perfusion with oxygenated Krebs-Ringer solution results in normal endothelial eccrine activity up to 1 h. Following this period there is substrate depletion but endothelial eccrine function can be demonstrated for up to 4 h. At 6 h this function cannot be demonstrated, suggesting degradation of the functional integrity of the endothelium.