This protocol is used to study the transport qualities of tight junctions. Using dilution potentials, you can measure the permselectivity and get an understanding of tight junctions in a tissue. This technique is specific and uses native tissues for functional studies of epithelia.
In addition, this technique measures the real-time physiological properties of a tissue, which is very useful. The biggest challenge is tissue preparation. Practicing the technique, as well as watching this video will help.
to confirm tissue viability is also an important step. To begin, place the desired segments of intestinal tissue collected from claudin-15 knockout mice into ice cold bubbled Ringer's solution and trim away the fat and connective tissue. After trimming away the fat and connective tissue, cut along the mesenteric attachments to open each segment longitudinally, then return the segments to the ice cold Ringer's solution and wash the pieces thoroughly.
To strip the muscle layer, pour two to three milliliters of fresh ice cold bubbled Ringer's solution into a silicone rubber-covered dissection plate under a dissection microscope and use the pins to secure the ends of one intestinal tissue segment in the dish with the mucosal side facing down. Using fine forceps, bluntly dissect the muscle layer from the underlying mucosa, being careful not to tear or introduce any holes into the tissue. Once the muscle layer has been removed, cut a piece large enough for a five millimeter diameter opening and place the tissue onto a piece of five millimeter punched filter paper wet with Ringer's solution mucosal side down using a black background to ensure that the opening in the filter paper is completely covered by the tissue without wrinkles.
To mount the intestinal preparations in an Ussing chamber, first remove any solution from the chamber before dissembling it. Lay the filter paper with the intestinal preparation mucosal side down on the mucosal side chamber using the black markings to align the chamber window with the hole in the filter paper. Carefully connect the serosal side chamber to the mucosal side chamber without moving the intestinal sheet.
Quickly refill both chambers with the HEPES buffer and place bubbling ones at the opposite end of each chamber away from the membrane. Reconnect the salt bridges and check whether the voltage is stable, then pulse the current to ensure that the connections are okay before allowing the system to equilibrate for about 15 minutes. To perform a dilution potential experiment, first wash both sides of the chamber with five milliliters of fresh pre-warmed HEPES buffer per side.
Turn on the recording system and set the range to 250 millivolts. Set the marker positions, set the recording system to measure and set the Ussing chamber systems to clamp mode. Once the measuring potential has stabilized, use the data for measurements.
Quickly replace the HEPES buffer from the mucosal side of the chamber with five milliliters of warmed dilution HEPES buffer supplemented with 75 millimolar sodium chloride. Once the membrane potential has peaked, replace the dilution buffer from the mucosal side with fresh HEPES buffer. To ensure that the tissue is viable, add 10 micromolar Forskolin to the serosal side.
Once the membrane potential difference has reached a peak and is beginning to decline, the experiment is over. To measure the transepithelial electrical conductance and baseline short circuit current, after washing both sides of the chamber as demonstrated, add five milliliters of fresh bubbled Ringer's solution to each side and set the range of the recording system to 2.5 volts. Set the marker positions, set the recording system to measure, and set the Ussing chamber system to clamp mode.
Once the short circuit current and conductance have stabilized, use the data for baseline measurements. To ensure that the tissue is viable, add Forskolin to the serosal side as demonstrated. Once the membrane potential difference has reached a peak and has started to decline, the experiment is over.
In this representative analysis, the baseline transmucosal conductance of the middle small intestinal segment was lower in the claudin-15 knockout mice under short circuit conditions compared to that observed in wild-type mice, while the baseline short circuit current was higher. Upon luminal sodium chloride dilution, a positive potential difference with respect to the serosal side was observed in wild-type mice, but the difference was decreased in the claudin-15 knockout mice. Similarly, the relative permeability of sodium chloride was also decreased in the knockout animals.
Calculation of the absolute permeabilities revealed that the absolute permeability of sodium decreased in the claudin-15 knockout mice, while the absolute permeability of chloride did not, suggesting that the decrease in the relative permeability is due to a decrease in the permeability of sodium. As expected, addition of Forskolin to the serosal side of the chamber did not cause a significant difference in the change of short circuit current between the knockout and wild-type mice. Since the intestinal segments demonstrated a large enough response to Forskolin, the membrane preparations were considered to be viable.
It is important to perform appropriate muscle layer removal and to ensure that tissue is viable. Please rinse the dissection section of the mice whether the preparation is viable.
