Animal studies and procedures were conducted according to the European Community regulations concerning the protection of experimental animals and the guidelines of the Regional Council of Braunschweig, Lower Saxony, Germany (File number 509.42502/09-01.03).
4.3.1 Experimental design
In order to study the effect of in vitro DON on the electrophysiological parameters across porcine small intestine, different in vitro DON concentrations were examined across different intestinal segments using Ussing chamber technique. The studies were done in 2 series; each had different in vitro DON concentrations (see table 6).
For assessment of the effect of oral DON on the respective parameters, pigs were fed on DON-contaminated wheat for about 5 weeks in the 2nd series (for compositions of experimental diet see table 5). For evaluation of the interaction effect between oral DON and LPS on the short circuit currents and tissue conductances, LPS was injected ip 3h before slaughtering of the animals in the 2nd experimental series (see table 6).
4.3.2 Animals
The animals used in both series are male castrated pigs. They were of the breed "Deutsches Edelschwein". The average body weight was approximately 24.3 kg in the 2nd series, at the
time of tissue preparation for the electrophysiological investigations. For the duration of the feeding experiments (~ 5 weeks) they were kept at the experimental facilities of the Institute of Nutrition, Friedrich Loeffler Institute Braunschweig, Germany, while the in vitro electrophysiological investigations using the Ussing-chamber technique were performed at the Department of Physiology, University of Veterinary Medicine, Hannover, Germany.
4.3.3 Chemicals
DON was purchased from (Sigma-Aldrich D-0156, München, Germany) diluted in isotonic saline. LPS was used from E. coli serotype 0111:B4 (Sigma-Aldrich L-2630, München, Germany) diluted in isotonic saline. The buffer solutions (Modified Krebs-Henseleit solution) that bathed the mucosal and serosal surfaces of the epithelial tissues consisted of (mmol/L):
After stunning, slaughtering and bleeding of the animals, the gastrointestinal tract was removed within 5 min after bleeding. In the first experimental series intestinal segments of about 80 cm length were immediately taken from the duodenum, mid jejunum and ileum. In the second series, segments from the mid jejunum were taken. The segments were immediately rinsed with ice-cold saline (0.9% NaCl) and kept in a modified glucose-containing Krebs-Henseleit buffer solution at 4°C, being continuously gassed with carbogen (95% O2, 5% CO2) until mounting in Ussing chambers.
4.3.5 Ussing Technique
After longitudinal incision along the mesenteric border, the intestinal segments were washed free of any remaining intestinal contents and the muscle and serosal layers were stripped off before mounting the mucosal layer into Ussing chambers to minimize the effect of the intrinsic neuromuscular system on the electrophysiological parameters. The chambers were
connected to glass circulation reservoirs through silicon tubes. The glass reservoirs were filled with 12.5 ml of modified Krebs-Henseleit buffer solution at both sides of the tissues and were adjusted at 38.4°C and gassed permanently with carbogen to maintain continuous circulation and the pH at 7.45. The electrophysiological experiments were carried out by computer controlled voltage / current clamps. The clamps were connected to the chambers through 3 M KCl-containing agar bridges and Ag/AgCl electrodes through which the short circuit currents were recorded. Fluid resistance and potentials were measured before mounting the tissue segments and corrected for during the experiments. For determination of electrophysiological parameters tissues were mounted in Ussing chambers with an exposed area of 1.13 cm2 with silicon rings and nets to prevent tissue damage and bulging. The tissues were clamped to 0 mV in order to eliminate the electrical gradients. Identical buffer solutions were used on both sides in order to abolish the chemical gradient. Thus the tissues were incubated in the absence of the transepithelial electrochemical gradient.
After an equilibration period (30 min), the basal values for potential differences, short circuit currents and electrical tissue conductances were recorded. DON was added to the mucosal side of the Ussing chambers at the previously mentioned concentrations (see table 6) to reach final volume of 13 ml in the glass reservoirs. An equal volume of modified glucose-containing Krebs-Henseleit buffer was added to the serosal side.
In order to assess the effects of DON and LPS on alanine and glucose transport, alanine (10 mM) was added to the mucosal side of the tissues in each Ussing chamber. Ten min later glucose (10 mM) was added to the luminal side. Responses of Isc after addition of alanine and glucose were recorded as differences between the constant values before addition and the maximal response (∆Isc).
4.3.6 Calculations and statistics
In both experimental series the short circuit currents (µEq.cm-2.h-1) and tissue conductances (mS.cm-2) were recorded via computer controlled voltage/current clamps. ∆Isc as well as ∆Gt were calculated for each animal for alanine and glucose as differences between the constant values before their addition and the maximal response after nutrients addition and the averages of ∆Isc and ∆Gt for each pig were calculated. The previous data were analysed using 2-factorial ANOVA (tissue and DON in vitro) and 3-factorial ANOVA (DON in vitro, ip LPS and DON in feed) including their interactions for first and second series, respectively and all
the results were presented as means, standard deviations and probabilities for the main effects and interactions.