Perfused Organs
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II.G.1
II.G.2 II.G.3
In Situ-perfused Isolated Intestinal Segments and Bile Secretion in Anaesthetized Rats Isolated Perfused Livers . . . . . . . . Isolated Perfused Kidneys . . . . . .
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II.G.1 In Situ-perfused Isolated Intestinal Segments and Bile Secretion in Anaesthetized Rats PURPOSE AND RATIONALE
The gut technique of in situ-perfused isolated intestinal segments in rats is used to determine the rates of intestinal absorption or intestinal secretion of a candidate compound, as well as in combination with a bile fistula to detect the hepatobiliary elimination of a candidate compound. By perfusion of intestinal segments (duodenum, proximal and distal part of the jejunum, and ileum) the site of intestinal absorption or secretion of a candidate compound can be determined. PROCEDURE
In general, rats are fasted overnight and are anesthetized with an intraperitoneal injection of 60 mg/kg pentobarbitone and anesthesia is maintained by a s.c. infusion of pentobarbitone at a rate of 20 mg/kg/h for the desired length of the perfusion experiment. Animals are laparotomized by a median incision and the common bile duct is cannulated with a polyethylene (PE) 10 catheter. Duodenum, or jejunal segments, or the ileum are cannulated for single pass perfusion or recirculated perfusion. After flushing the lumen of the respective intestinal segment with 40 ml of medium, perfusion is performed in situ at a very low continuous flow rate of 0.5 ml/min, ensuring a minimal perfusion pressure. The candidate compound is added to the perfusion medium at an appropriate concentration to reach detectable compound levels in blood (plasma) and bile. Body temperature and perfusion medium are maintained at 37 °C. Bile aliquots are collected at 10 to 30 min intervals. Blood samples (100 µl) are collected
from the jugular vein every 30 minutes in EDTA tubes. At the end of the perfusion experiment the livers are removed from all animals, total weight is determined and the livers are immediately frozen for determination of tissue level of the compound in liquid nitrogen and then stored at –20 °C until analysis. For analysis of tissue levels of the candidate compound the frozen livers are homogenized in water (1 g tissue in 1 ml water (or solvent to extract the candidate compound from the tissue)) using an ultra-turrax. A continuous bile flow throughout the experiment is the best measure of the functionally integrity of the organ (typically bile flow are shown in Fig. 1). Animals with an initial bile flow below 100 µl/30 minutes should be excluded from the final evaluation of the data. EVALUATION
The concentrations of the compound are determined using appropriate analytical methods for the respective candidate compound in the perfusate at the end of the perfusion experiment, plasma, bile and hepatic tissue. From the data the site of intestinal absorption using different perfused intestinal segments in separate experiments as well as the degree of hepatobiliary elimination can be estimated. Appropriate analytical methods with suffici
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