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2 (5%) in oxygen at 37°C. The transmural PD was monitored with a pair of calomel electrodes. Two types of Ussing chambers were used. In one type, the electrical field of the voltage clamp was perpendicular to the intestinal segment, which made it possible to estimate tissue resistance in absolute terms by using an automatic voltage damp device to measure SCC. PD and SCC were recorded on a polygraph. Values of PD and SCC reported in the text, tables, and figures are given as the mean ± SEM. After mounting the tissue, the PD was allowed to stabilize for about 30 min. In control experiments, PD and SCC remained constant in infected and noninfected animals after this stabilizing period (24). For testing of drugs, each tissue was exposed to one drug in increasing concentrations with a 10-min interval between administrations. The drugs used were administered in volumes of 20 μl. At the end of the experiments, 200 μl of theophylline solution was added to the mucosal and serosal solutions to a final concentration of 1 mM to test tissue viability. If theophylline increased the PD by less than 40% of the control value, the experiment was discarded.
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20
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0343312318
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data not shown
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O. Lundgren et al., data not shown.
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21
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0343747994
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note
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-1) for at least 24 hours before use. In control experiments this procedure prevented any adsorbance of the tracer to the plastic tubing. After placing the segment in the organ bath, the intestine was perfused for 20 min without sampling. Then 20-min samples were collected in tubes at the outflow end of the perfusion system. In one series of experiments (Fig. 2), in which the viability of the intestinal preparation was tested, the perfusion fluid was sampled during three consecutive 20-min periods. For reasons given in the text, only two samplings were performed in the experiments with drugs. Drugs were administered to the organ bath between the first and second sampling period. Radioactivity in duplicate samples was measured from the inflow and outflow solutions. The sample (100 μl) was mixed with 3.0 ml of scintillation fluid (Ultima Gold XR, Packard). At least 10,000 decays were measured in a Packard scintillation counter (Packard 1900 TR Liquid scintillation analyzer). Net fluid transport was estimated from the rate of perfusion, and the radioactivity of the solutions entering and leaving the intestine was determined (26). The transmural PD of the intestinal segment was recorded by two electrodes. One electrode was connected by a T-tube to the inlet of the perfusion system and the other was in contact with the solution in the organ bath. The PD was continuously recorded on a polygraph.
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22
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0343312317
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note
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Diarrhea in the mice was judged by a scoring system, with a score of 1 indicating unusually loose yellow stool and a score of 4 a completely loose stool A score of ≥2 (mucous with liquid stool, some loose but solid stool) was considered to indicate diarrhea. The scoring was performed by the same individual twice daily after administration of the rotavirus.
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24
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Supported by grants 28255 and 10392 from the Swedish Medical Research Council, and by the Knut and Alice Wallenberg Foundation.
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