Library

Notes: Introduction:  The pathomechanisms of bovine laminitis are still poorly understood. Isolated perfused organ models are promising tools to study the biological mechanisms that lie behind diseases. They are preferable for ethical and financial reasons but also for experimental necessities.Aims:  To develop an isolated haemoperfused distal cow limb model, basing on an already existing porcine limb model used in experiments studying the effects of bioactive molecules.Methods:  Isolated limbs and blood were obtained from routinely slaughtered cows and subsequently perfused in the laboratory of a biotech company (Vitro-Tec, Berlin) with expertise in organ perfusion. A standard perfusion apparatus enables perfusion for up to six hours under close to physiological conditions. Perfusion pressure and flow were calculated based on values available for horses and pigs. The oxygen saturation was kept at 100% and the pH between 7.35 and 7.45. Glucose was added as nutrient. The lactate concentration was measured as a parameter for oxygen supply and free haemoglobin as an indicator for haemolysis. Potassium content and pressure served as vitality indicators. Additionally, morphological criteria of tissue integrity were assessed using light and electron microscopy.Results:  A constant flow of 150–200 ml/min was optimal. The resulting pressure ranged from 30 to 117 mmHg. Light microscopy revealed, with exception of a few thromboses, no cellular damage after 4 h. The perfusion was successfully extended to 5 h. Then first pressure related alterations including vascular dilatations and cellular degeneration occurred in the dermal lamellae but not in the other regions. Potassium never exceeded physiological values.Conclusion:  The model is now available for experiments. Equipment and technical implementation have been standardized and described in a standard operating procedure. The model will be challenged by bioactive molecules such as lactate, neuropeptides and chemokines. Ischaemic conditions will be simulated and studied.This work was supported by the European Communities under the Lame Cow Project QLK5-CT-2002-00969.The authors are solely responsible and the work does not necessarily represent the opinion of the European Communities.