The method we've developed here has turned out to be extremely useful in our work, trying to find out the molecular mechanisms of secretion of hormones from the gut, but also absorption of nutrients, and the coupling between the two. The main advantage of this highly physiological technique is that the gut is left in situ, while allowing detailed studies that are usually only performed within In Vitro models. First place the rat on a heated operating table, confirm a lack of response to toe pinch, and make a skin and peritoneal excision to expose the abdominal cavity.
Move the small intestine to the side to expose the terminal region of the colon. And ligate the supplying vasculature to the colon. Gradually extract the colon, starting from the terminal region, and moving towards the small intestine, hydrating the tissue with isotonic saline, and using cotton swabs to remove the connective tissue as necessary.
When all of the colon has been removed, insert a 15 centimeter piece of plastic tubing into the proximal end of the small intestinal lumen, and secure the tubing with sutures. Then use a 10-milliliter syringe and a syringe pump to carefully flush the small intestine with room temperature isotonic saline at a 25-milliliter per minute flow rate. When the tissue has been emptied of chyme, adjust the tissues so that the upper mesenteric artery is accessible and remove the connective tissue and fat tissue to expose the artery.
Use fine point forceps to place two sutures under the mesenteric artery and two sutures under the portal vein. Fill a plastic 22-gauge catheter attached to a rolling pump with perfusion buffer and use a pair of surgical scissors to cut a small hole in the mesenteric artery, immediately inserting the catheter into the incision. Insert the catheter within two minutes of cutting a hole in the artery, otherwise the gut will likely suffer from ischemic damage.
As soon as the catheter has been secured with a suture, start the rolling pump to initiate perfusion at a 7.5 milliliter per minute flow rate. When the blood vessels in the gut and portal vein turn pale, cut a hole in the portal vein. Insert a metal catheter, and secure the metal catheter with another suture.
Take care when inserting the catheter into the portal vein, as the vein is thin walled. However, as the guard is now being perfused, quick insertion is not crucial. After collecting the perfusion output for one minute, measure the volume and click execute experiment in the pressure recording program to initiate the perfusion pressure acquisition recordings.
Then cover the gut with a moistened lab tissue to keep it from drying out, and confirm that the distal end of the intestine is not blocked, and a luminal haploid can exit. To measure the partial pressure of oxygen and carbon dioxide, collect perfusion buffer through a three-way stop-cock valve immediately before it enters the organ, after approximately three minutes of perfusion, and from the catheter inserted into the portal vein. Place the samples on ice, and use an automated blood gas analyzer to measure the samples soon after collection.
Use a fraction collector to obtain the first baseline samples. After 10 to 15 minutes of baseline collection, use a syringe pump to administer the first intraarterial test stimulant through a three-way stop-cock at a 35 milliliters per minute flow rate. Perform luminal stimulations by an initial stimulation boas injection delivered at a 2.5 milliliter per minute flow rate over the first five minutes of the stimulation, followed by administration of the test solution at 5 milliliter per minute flow rate.
As soon as a luminal stimulation period is over, flush the lumen with isotonic saline at a 2.5 milliliter per minute flow rate for five minutes. Then collect baseline samples at 5 milliliters per minute for 15 to 30 minutes before the next test substance is administered. At the end of the experiment, administer a suitable, positive control to test for the responsiveness of the preparation for five to 10 minutes, collecting and additional 10 to 15 minutes of baseline samples at the end of the positive control stimulation period.
Here is shown an example of good quality data, demonstrating Glucagon-like peptide-1 or GLP-1, secretion from an isolated perfused rat small intestine collected at one minute intervals. Under basal conditions, the secretion was steady while both before and after administration of the test stimulus and the positive control, a robust secretory response was evident. These GLP-1 secretion data obtained from an isolated perfused rat small intestine are of poor quality with an unsteady secretion measured at basal conditions that drifted upward throughout the testing in a manner that appeared to be unrelated to the test substance administrations or to the positive control.
It is therefore impossible to conclude whether the increased GLP-1 secretion observed at the end of the vascular fructose stimulation was a fructose mediated response. While attempting this procedure to care to secure the catheters properly and to thoroughly clean the perfusion equipment as the perfusion buffer provides an excellent medium for bacterial growth. Following this procedure, other methods like In Vitro studies on hormone-secreting cell lines of primary intestinal cell cultures, can be performed to directly investigate the intracellular production of cyclic AMP or intracellular calcium.
So we've already used the method now for elucidation of a number of processes behind absorption of nutrients and the mechanism of secretion of the hormones and it has turned out to be extremely useful. Don't forget that working with blockers or activators of molecular sites can be extremely hazardous and precautions such as using a lab coat, gloves, and safety glasses, a fume hood should always be taken while performing this procedure.
