The overall goal of this technique is to identify changes in the neural regulation of colonic motility in mice using video imaging. This technique can help answer key questions related to the enteric neuroscience field, such as do subtle genetic mutations in neuronal genes affect colonic motility. The main advantage of this technique is that the tissue is a cyst in the absence of central nervous system input, and as a result the method allows investigation of the enteric nervous system's involvement in the control of gastrointestinal motility.
This technique extends towards the therapy of neurodevelopmental diseases such as autism, as gene mutations altering synaptic function in the brain, might also alter gut motility. Begin by using dissecting forceps and scissors to make an incision through the epidermis overlying the lower abdominal muscle layers. Open the abdominal cavity along the midline to the sternum.
Moisten the tissue with physiological saline and use the forceps to gently lift the cecum four to five centimeters above the abdomen. Then, using fine dissection scissors, trim the mesentery, taking care not to handle, stretch, or cut the gastrointestinal tissue. Remove the bladder, testes, and any other excess tissue.
Then, using coarse dissecting scissors, make two vertical incisions approximately 0.5 centimeters from the midline into the pelvic bone on each side of the colon, and use the fine scissors to separate the rectum from the adjoining pelvic tissue. Trim the mesentery from the colon and rinse the full-length colon tissue in a beaker containing physiological saline. To remove the fecal pellets and other intestinal contents, first trim approximately one centimeter from the end of a 200 microliter pipette and attach the pipette to a five milliliter syringe filled with physiological saline.
Then, after removing the cecum and the rectum, place an insect pin in the most proximal end of the colon where the striations in the mucosa are visible to orient the tissue. Now, gently grip the tissue at the proximal end, and insert the pipette tip into the lumen and flush the saline through the colon into a waste container. Next, with the same syringe and pipette tip, flush any debris from the tubing attached to a two-chambered organ bath with fresh physiological saline.
Set the organ bath so that the chambers will be continuously superfused with physiological saline bubbled with carbogen and use a temperature probe to regularly confirm that the saline temperature remains between 33 and 37 degrees Celsius. Use a rubber stopper with a five millimeter inside diameter glass tube inserted through the center to maintain the pressure in the inflow reservoir. Then, fully submerge a five to seven centimeter piece of colon into the organ bath chamber and use forceps and standard cotton sewing thread to cannulate the proximal end of the colon to the inlet tube and the distal end of the colon to the outlet tube.
After determining the baseline distance between the proximal and distal cannulations of the colon, equilibrate the tissue in physiological saline for 30 minutes. To record the intestinal movements, while the colon is acclimatizing, position a video camera 10 to 15 centimeters above the organ bath on a standard laboratory retort stand, and set up the appropriate video capture software. When the camera is ready, record four 15 minute video segments of the colon under control conditions.
Then, superfuse the drug of interest into the organ bath for 30 to 60 minutes, capturing another four 15 minute video segments. Finally, replace the treatment solution with fresh physiological saline and record four more 15 minute video segments during the one-hour wash out period. Spatiotemporal maps of the colon, in response to the different treatment periods can then be generated.
To assess whether synaptic mutations alter colonic migrating motor complexes, or CMMC's, when the enteric nervous system is pharmacologically perturbed, in this experiment, the five HT3 and 4 receptor antagonists, tropisetron, was added to the colon preparation organ bath chamber. In the presence of tropisetron, NL3R451C mice exhibited a decrease in CMMC frequency compared to their wild type litter mates. Although no significant differences were observed between the wild type and NL3R451C contractility during controlled conditions, tropisetron significantly reduced the CMMC frequency in both wild type and NL3R451C mice.
Moreover, tropisetron imposed a larger effect on the frequency of CMMC's in NL3R451C mice compared to wild type animals. Once mastered, this technique can be completed in four hours if it's performed properly. While attempting this technique, make sure that the organ bath is maintained at a constant temperature, it has a continuous supply of carbogen, and overflow of physiological saline is prevented by a vacuum.
Following this procedure, other techniques such as immunocytochemistry can be performed to answer additional questions such as are there changes in neuronal proportions or cell numbers in mice exhibiting altered colonic motility? Since its development by this method has allowed a variety of studies of intestinal motility in guinea pig and rabbit, rat, and other species, and most particularly in this case in the mouse. After watching this video, you should have a good understanding of how to identify changes in the neuronal regulation of colonic motility using video imaging.
