The gut-blood barrier is a multilayer system that controls the passage of nutrients, bacterial metabolites, drugs, and other exogenous compounds from intestinal lumen to the blood stream. As we know, the integrity of the barrier may be impaired in gastrointestinal as well as in cardiovascular and some metabolic diseases which may result in easier access of biologically active compounds such as gut bacterial metabolites to the blood stream and may affect functioning of entire organism. Therefore the permeability of the gut-blood barrier may be a marker of both intestinal and extraintestinal diseases.
In this video, we present a direct measurement of the gut-blood barrier permeability in rats using portal blood sampling after administration of a test substance to the digestive tract. We describe in detail the categorization of the portal vein and inferior vena cava just above the hepatic vein confluence. Blood sampling from portal vein and inferior vena cava allows to evaluate not only the gut-blood barrier permeability but also a hepatic clearance and absorption pathway of molecules of interest such as gut bacteria metabolites or medicines.
This method is virtually not influenced by liver and kidney function and preserves the intestinal blood flow which is a strong advantage over commonly used in vivo and in vitro methods. Animals should be fasted overnight before the procedure. Perform all procedures during general anesthesia.
Animals are euthanized after the experiment. Our procedure starts with the insertion of the line for intraintestinal administration. Here we propose intracolonic administration of marker.
It maybe be modified by administration at various levels of digestive tract. For example, stomach or duodenum. However, take into account the variable speed of peristalsis and possible interactions with enzymes and gastric acids while administering a marker into upper parts of the gastrointestinal tract.
Use pediatric Foley catheter size 10 F or 8 F as a colonic catheter. Mark the catheter to indicate the part which will be inserted into the colon approximately eight centimeters. Check the anal region and the count of the stools in the rectum before inserting the catheter in the colon.
If the stools are present, empty the rectum by massaging the rectal area. Put a lubricant along the catheter. Next, moisten the anus and its surroundings with a lubricant.
Insert the catheter with a guide wire through the external anal sphincter. Make slow forward, backward and circular movements. Keep on checking the location of the catheter by abdominal palpation while inserting the catheter.
Next step is the inferior vena cava catheterization. You can skip this procedure in gut permeability studies. Try to feel the pulse on the femoral artery and cut the skin longitudinally for the length of about two centimeters in the place where the pulse is palpable.
Dissect the fascia and muscles to visualize the neurovascular bundle. Next, dissect the femoral vein from the neurovascular bundle. First nerves, then the femoral artery and then the vein.
Be careful during the dissection of the bundle since several tiny branches of the femoral vein may easily be damaged. Next put two ligatures on the femoral vein. Do not tie the knots yet.
Catch the ends of the proximal ligature with a needle holder. Then carefully pull the ligature ends with the holder upwards to close the proximal part of the vein. Wait until the vein is filled with blood and tie the distal knot.
Next, make a small incision on the vein between the knot and proximal ligature using microsurgical scissors. Then insert the catheter using tweezers or a needle with a curved end as we do in the video. Puncture the vein and use the bent tip of the needle as a guide for the catheter.
Loosen the proximal ligature while inserting the catheter. Insert the catheter for approximately six to seven centimeters depending on the size of an animal to place the proximal tip of the catheter in the inferior vena cava just above the hepatic vein confluence. The proper placement of the catheter should be confirmed after the experiment.
Secure the catheter in the femoral vein with two single surgical knots. Tie the proximal ligature as well. Check the patency of the catheter by attempting to draw blood with a syringe.
Rinse the catheter with a 0.3 milliliters of the heparinized saline. Close the surgical wound with two layers of single stitches. Now we move on to portal vein catheterization.
Prepare the portal catheter. It consists of a needle, flexible polyurethane catheter, flexible polyethylene tip of the catheter, plug, and the ligature. First step of the procedure is performing a midline laparotomy.
After preparing the operating field, cut the skin longitudinally from the xiphoid of the sternum to the navel. Cut the muscles of the abdominal wall along the white line. Next, expand the cut rostrally in the y shape so that the xiphoid cartilage is between two cuts.
Next step is the portal vein dissection. First, moisten the swabs with saline. And then exteriorize the cecum, ascending and transverse colon, and small intestine loop.
Put the intestines on the left side to expose the root of the mesentery. Cover the intestines with gauze moistened with a physiological saline to protect them from drying. To expose the portal vein move carefully the hepatic lobes to the sides or upwards towards the diaphragm with the moistened swabs.
Here we can see the portal vein and upper mesenteric vein. Localize the part of the portal vein that is not covered with the mesentery and pass the ligature under the portal vein. To protect tissues from damage, moisten the ligature with a physiological saline solution.
Clamp with forceps the ends of ligature and tighten it gently to stabilize the vessel. Pass the longer part of the catheter's ligature under the free part of the portal vein and pull it so that the catheter is located just next to the portal vein. Insert the needle into the upper mesenteric vein three millimeters below the junction of upper mesenteric vein and portal vein.
Hold the needle at a 30 degree angle and after entering into the vein reduce the angle and advance the needle almost horizontally in parallel to the portal vein for a length of approximately six to seven millimeters. Apply one or two drops of tissue glue at the place where the needle is inserted. Remove the swabs that covered the liver.
Afterwards put the intestines back to the abdominal cavity. Moisten the intestines with a warmed up saline solution and cover it with a moistened sterile gauze. Check the patency of the catheter and rinse the catheter with heparinized saline.
Venous blood spontaneously backflows in the catheter. After five minutes check the color of the intestines and peristaltic movements to make sure that the proper mesenteric blood flow is maintained. In the end, close the abdominal cavity with three layers of stitches exteriorizing the distal part of the catheter around the navel.
To collect blood from the portal vein, open the portal catheter plug and let the blood flow freely. Use a syringe and a blunt needle and collect no more than 0.7 milliliters of blood. Rinse the catheter with heparinized saline and close the catheter plug.
Here we present exemplary portal blood sampling protocols for gut permeability assessment. Blood samples from the inferior vena cava are necessary for hepatic clearance and absorption studies. To collect blood from the inferior vena cava proceed exactly as when taking blood from the portal vein.
Here we present an exemplary blood sampling protocol for liver clearance measurement and for tracking the gut-portal blood-liver-systemic blood pathway of selected compounds. In our studies we used a bacterial metabolite, trimethylamine as a marker of permeability. However many other substances including class permeability markers may be used as well.
Various bacteria and metabolites and tracts may be also used to tract the absorption pathway or hepatic clearance. Remove the guidewire and inflate the balloon using adequate volume of sterile water usually one milliliter but check actually balloon size before insertion. The balloon diameter should not exceed one centimeter.
Place the rat head down to minimize the risk of the outflow of the administered solution from the colon. Slowly administer the tested substance using drainage port and colonic catheter. We used trimethylamine in a dose of 100 milligrams per kilogram body weight dissolved in 0.7 milliliters of saline.
After ten minutes deflate the catheter balloon. Hepatic clearance understood as a hepatic extraction may be expressed by the difference between portal blood concentration and inferior vena cava blood concentration or by the ratio of inferior vena cava and portal blood concentration. We have successfully measured the gut-blood barrier permeability and liver clearance of trimethylamine in rats.
Here are the representative results which demonstrate that hypertensive rats have increased colon permeability to TMA in comparison to normotensive rats. In another research we studied whether high salt intake affects the gut-blood barrier permeability and liver clearance of trimethylamine. Intracolonic administration of trimethylamine produced a significant increase in portal blood TMA concentration.
The size of the increase and hepatic clearance were similar between the groups. We also tracked the absorption pathway of short chain fatty acids. We measured the concentration in stool, portal blood and peripheral blood.
In this video we described a comprehensive protocol that includes direct measurement of the gut-blood barrier permeability in vivo which, in contrast to commonly used methods, is not affected by liver and kidney functions, hepatic clearance measurement, and third, evaluation of the absorption pathway of exogenous compounds. The presented technique may be adjusted to other experimental purposes. For example, portal blood pressure measurement or drug administration directly to the portal vein.
