3.3.1 Chemicals and solutions for the electrophysiological experiments
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). The buffer solutions (Modified Krebs-Henseleit solution) that bathed the mucosal and serosal surfaces of the epithelial tissues are shown in table 6. All solutions were prepared with Indomethacin (10-5M) to reduce prostaglandin synthesis and were adjusted to an osmolality of 300 mosmol/kg with mannitol to maintain the same osmotic pressure in both sides of the chambers. The PH of the buffer was adjusted to 7.45 using HCl. The buffers were continuously gassed with carbogen (95% O2, 5% CO2) at 38.4°C all over the experiment.
Glucose was added to the serosal buffer to provide an energy substrate and achieve active transport conditions.
Table 7: Compositions of Krebs-Henseleit buffer solution for mucosal and serosal sides
Mucosal buffer (mmol/L)
Serosal buffer (mmol/L)
NaCl 113.6 113.6
KCl 5.4 5.4
HCl 0.25 0.4
MgCl2.6 H2O 1.2 1.2
CaCl2.2 H2O 1.2 1.2
NaHCO3 21.0 21.0
Na2HPO4.2 H2O 1.2 1.2 NaH2PO4.H2O 0.3 0.3
mannitol 31.96 23
glucose - 10
3.3.2 Tissue sampling and preparation
The animals were slaughtered by stunning, bleeding for about 2 min during which blood samples were collected from each pig in two Lithium-Heparin tubes. 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.
Duodenum was about 20 cm after pylorus, mid jejunum was 3 meters after pylorus and ileum was one meter proximal to ileo-caecal valve. In the second series segments from the mid jejunum were taken (the fourth meter after pylorus). 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. 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 by scraping the serosal surface with the edge of a glass slide before mounting the mucosal layer into Ussing chambers keeping the tissues wet by applying ice-cold buffer.
The blood samples were centrifuged for 10 minutes at 3600 rpm and 4°C and plasma was separated in four Eppendorf tubes (2 ml each) for each pig and stored at -20°C until analysis.
3.3.3 Ussing Chamber Technique
The aim of this study was to estimate the effects of DON on the electrophysiological parameters across the porcine small intestines in vitro. The electrogenic transport of glucose and alanine were assessed and used as indicators for such transport processes. These nutrients are co-transported with sodium ions across the mucosal surface of the epithelial cells via specific transporters. The tissue conductance (Gt) represents the reciprocal of the electrical tissue resistance and considered as a useful measure of the integrity of the intestinal barrier including the transcellular and paracellular pathways. The physiological approach that was used to investigate the previous parameters was Ussing chamber technique. It is a valuable method used for measuring nutrient transport across epithelial tissues (CLARKE 2009). The chambers are made of two acrylic glass fragments and were connected to glass circulation reservoirs through silicon tubes (Figure 10). The glass reservoirs were filled with 12.5 ml of modified Krebs-Henseleit buffer solution at both sides of the tissues and were water jacketed to enable warming of the intestinal preparation at 38.4°C and gassed permanently with carbogen (95% O2, 5%CO2) to maintain continuous circulation and the pH at 7.45. The electrophysiological experiments were carried out by computer controlled voltage/current clamps (scientific instrument, Dipl.-Ing. K. Mußler, Aachen, www.Kmsci.de). The clamps were connected to the chambers through 3M KCl-containing agar bridges and Ag/AgCl electrodes (Mettler Toledo Prozessanalytik GmbH, Germany) 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. To apply the standard experiment, the electrical and chemical gradients must be abolished. This was achieved by operating the chambers with the buffers in the absence of the intestinal preparations. During this period the bubbles were removed and the buffer resistance was compensated. The adaptation time for the buffers was about 10 min after which the buffers reached the desired temperature 38.4°C.
Clamping the voltage to 0 mV eliminate the electrical gradient.
For determination of electrophysiological parameters, the tissue preparations were mounted in Ussing chambers with the mucosal surface upward and with an exposed surface area of 1.13
cm2 with silicon rings and nets to prevent tissue damage and bulging. The tissues were allowed to adapt and recover under open circuit conditions for 10-20 minutes and were then 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.
Figure 10: A schematic diagram of Ussing chamber
After an equilibration period of about 30 minutes, the basal values for potential differences, short circuit currents (Isc) and electrical tissue conductances (Gt) were recorded automatically. DON was added to the mucosal side of the Ussing chambers at the previously mentioned concentrations (Table 6) to reach a 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. The experiment was ended 20
minutes after addition of glucose. The Isc and Gt values for alanine and glucose were recorded automatically and the responses of Isc and Gt after addition of alanine and glucose were calculated as differences between the constant values before nutrient addition and the maximal response (∆ Isc and ∆ Gt).
3.3.4 Calculations and Statistics
The differences between the basal values before addition of the nutrient and the maximal response were calculated using Microsoft Office Excel 2003 and represent ∆ Isc and ∆ Gt.
The previous data are analysed using 2- and 3-factorial analysis of variance (ANOVA) and presented as means ± standard deviation (SD) whereas n represents the number of pigs and p-values < 0.05 were considered to be significant.