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The first part of this dissertation deals with the standardisation of the isolated perfused porcine limb and the characterisation of its metabolism.For this pur pose, two test groups (group 1.1.2 and 1.2) were established, which differ with regards to glucose application over a time period of 7 hours. It turned out that by glucose application on an hourly basis (1 g glucose added to the dialysate),a physiological glucose level in the perfusate was maintained. In contrast, glucose application every three hours (3 g glucose added to the dialysate) resultedin large positive and negative deviations from physiological values. The arterio-venous glucose difference as a key parameter for glucose uptake by the cells was not altered after insulin application. The metabolic status of the model was characterised by comparing two test groups (group 1.1.1: 100 mI/min perfusion flow, oxygenation with 100 % air, group 2: 230 - 250 mI/min perfusion flow, oxygenation with an air-oxygen mixture). No difference between these two groups with regard to oxygen consumption was observed. Overall, oxygen consumption varied between 0.04 and 0.1 mI/min*100 g organ weight, which was below the values for skeletal muscle reported in the literature (0.18 ± 0.04 mI/min*100 g weight). Tissueedema formation was evaluated by calculating the percentage increase in weight,which was found to be 2-10 % over a time period of 7 hours. This was in agreement with literature data (6 % ± 4).To investigate the percentage of aerobic vs. anaerobic metabolism, the lactate/pyruvate ratio was determined in the second group. The ratio was found to be considerably higher (<80) compared to in vivo results for piglets from the literature (13.5 ± 3.4). Additionally, glycogen content
served as a parameter since it is decomposed under hypoxic conditions. It was found in both groups that muscle tissue did not contain any glycogen after 7 hours (with and without insulin application) as shown by PAS staining. Judged from additional parameters like organ resistance (the ratio of perfusion pressure in mmHg and perfusion flow in mI/min) and lactate content, which were considerably higher in the first (low flow) group oxygen supply seems to be critical. The observations clearly show that there is an anaerobic metabolism in the isolated porcine limb.Oxygen deficiency during the perfusion period of 7 hours might be caused not only by low flow, but also by a decreased number of red blood cells available for oxygen transport. Hemolysis was determined as potassium and hemoglobin release from the erythrocyte. The potassium concentration in the perfusate of group 1.1.1 was within the physiological range except the last hour of testing, whereas the concentration in the 2 nd group was outside the range starting from the 4 Ih hour. Also the concentration of free hemoglobine was considerably higher in group 2 compared to group 1.1.1. These results demonstrate that the higher perfusion flow of group 2 resulted in a higher hemolysis.A histological examination of the skin (Light Microscopy) and the skeletal muscle (Electron Microscopy) was carried out after the characterisation and standardisation procedure was finished. The skin samples were intact to a great extent before and after the testing period except edematic loss of connective tissue. In contrast, the muscle samples were remarkably different. The samples taken immediately after the pig was killed showed quite a lot of intact cells and mitochondria, whereas those taken from deeper stratums and the surface after the test showed degeneration of mitochondria and other cell organells. This surely indicates a lack of oxygen supply, b
ecause especiaIly mitochondria are extremely sensitive for oxygen deficiency.In the second part of this work the optimised isolated perfused porcine limb was used as a transdermal resorption test system. The test substance, Nitroglycerin (NG) was applied onto the skin as a transdermal therapeutic system (TTS) in two different concentrations (TTS 5: 25 mg; TTS 10: 50 mg). It was shown that - starting from hour 3 - NG detectable in the perfusate was above the therapeutic level (1 ng/ml). The NG concentrations determined were considerably higher in the testing organs (TTS 5: up to 17 nmol/l, TTS 10: up to 20 nmol/l) compared to humans (TTS 5:up to 1.2 nmol/l, TTS 10: up to 5 nmol/I).However, the release of NG from TTS 5 was detectable one hour later compared to TTS 10 although the NG concentration detectable after 5hr in the dialysate was not different between these two
groups (TTS 5 and TTS 10). The concentrations observed for both groups in the dialysate were 50 % lower than the corresponding values in the perfusate. Until the last hour the concentrations were almost constant, but increased afterwards. The analysis of NG in control samples was negative at all times.In conclusion, the test conditions using the isolated perfused porcine limb as a model for transdermal resorption were improved remarkably by increasing the perfusion flow and oxygen supply. The fact that NG was detectable in the perfusate after applicationas TTS is proving that this organ model might be useful as a test system in pharmacological research and development.