The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The overall goal of this procedure is to evaluate the rate and extent of transport of lipids and drugs into the intestinal lymphatic system following intestinal delivery. This is accomplished by first isolating and cannulating the mesenteric lymph duct for lymph collection. In the second step, the duodenum is cannulated to enable infusion into the intestine, and then the carotid artery is cannulated to enable the collection of blood samples.

In the final step, the experimental treatment is delivered into the duodenum and the lymph and blood are collected for the quantification of systemic lipid or drug levels. Ultimately, this model can be used to evaluate the total absorption and intestinal lymphatic transport of lipids and drugs following intestinal delivery. The main advantage of this technique is that it allows the cannulation of the mesenteric lymph duct directly and collection of lymph across the entire experimental period.

This allows an estimation of the absolute massive material that flows into and through the mesenteric lymph. In vivo, Though this model enables the in vivo evaluation of drug and lipid transport. It can also be used to measure the intestinal lymphatic transport of any factor of interest, as well as the change in lymphatic transport of such factors in response to different challenges or disease states.

Visual demonstration of this method is critical as the cannulation of the mesenteric lymph duct is complicated and difficult by the fact that the duct is small, fragile, and in fasted animals. Transparent Demonstrating the procedure will be my colleague and fellow faculty member here at Monash University, Dr.Natalie cus, and a senior postdoc in the group, Dr.LJ Hugh, On the day before the surgery, cut the cannulas to the appropriate lengths and use a sterile surgical blade to make a bevel at the tip of each piece of tubing. Next, to prepare the intra duodenal cannula, loop the cannula back onto itself and then heat the cannula to create an anchor point in the tubing.

On the day of the surgery, shave the fur from the right side of the abdomen and neck and left clavicle regions of an anesthetized adult rat, and then clean the surgical regions aseptically with three successive povidone iodine solution and 70%ethanol scrubs to cannulate lymph duct. Place the animal with its right side facing up and use a sterile scalpel blade to open the top layer of the abdominal muscle wall with a straight four centimeter incision extending from the xiphoid process to the right flank approximately two centimeters below the costal margin. Retract the small intestine under the left abdominal wall and use two to three pieces of sterile gauze saturated with normal saline to keep it in place.

Then bridge the rat over a 10 milliliter plastic syringe placed horizontally under the animal's back at the level of the right kidney. To assist in the visualization of the mesenteric lymph duct, locate the superior mesenteric lymph duct, which is approximately 0.5 to one millimeter in diameter, perpendicular to the right kidney, and immediately rostral and parallel to the dark red pulsating mesenteric artery. Note that in non fasted rats as shown here, the superior mesenteric lymph duct is white, opaque and easier to visualize than infested rats.

Next, pass a pair of straight forceps through the perineal fat bed at the lower margin of the right kidney parallel to the superior mesenteric lymph duct and through the connective tissue layers immediately below the vena cava. Then using the tip of the forceps, pull the lymph cannula through the perren fat bed with one end immediately adjacent to the mesenteric lymph duct and the other exteriorized from the animal at the level of the right kidney. Then using a surgical microscope and jewelers forceps, isolate them mesenteric lymph duct through the overlying layers of connective and fat tissue by blunt dissection starting at the lower end and taking care not to damage the lymph duct when the duct has been isolated.

Use the tip of the fine tip forceps to make a small hole in the lymph duct. After ensuring that the lymph cannula is completely filled with anticoagulant solution and no air gaps, use a small pair of forceps to insert the lymph cannula approximately two to four millimeters inside the mesenteric lymph duct via the puncture. If the cannulation is successful, a gradual flow of intestinal lymph will be observed from the free collecting end of the cannula.

When this occurs, place a small drop of cyanoacrylate glue over the entrance hole into the lymph duct to secure the cannula, taking care not to occlude the vessel with excess glue to cannulate the duodenum. First, locate the tissue as the bright pink section of the small intestine with a upon gentle downward pulling, reveals the stomach. Then use a sterile 23 gauge needle to make a small puncture hole in the duodenum at the pylori, approximately two centimeters below the junction of the stomach and duodenum.

Next, insert the js shaped hook end of the duodenal cannula through the puncture and secure the cannula in place with a drop of Sano acrylate glue. Then use sutures to close the abdominal muscle wall incision And the carotid artery cannulation can be performed before or after the cannulation of the mesenteric lymph duct. We typically prefer to cannulate the carotid artery before the cannulation of the lymph duct, so to reduce the potential of dislodging the lymph cannula when moving the animal To cannulate the carotid artery, first, mark the carotid artery cannula 2.5 centimeters from the bevel tip to identify the portion of the tubing to insert into the artery.

Then connect the un beveled end of the cannula to a 25 gauge needle attached to a syringe filled with anticoagulant solution and fill the cannula with the solution, making sure no air gaps are present. Next, make a one to 1.5 centimeter longitudinal incision through the skin layer above the left of the trachea in the sagittal plane, and then use blunt tip forceps to dissect the subcutaneous connective tissue and expose the underlying paired neck muscles. Retract the neck muscles to reveal the pulsating left carotid artery located approximately one centimeter below the skin surface.

Then clean the overlying connective tissue from the artery by blunt dissection and use blunt tissue forceps to isolate an approximate one centimeter section of the artery. Next, use fine tip forceps to thread two silk sutures underneath the carotid artery, placing them at each end of the isolated artery section. Then tie off the suture furthest from the rat's heart and clavicle, and place a pair of straight fine tip forceps under the carotid to occlude the blood flow through the artery.

Now using fine tip iris scissors, make a small incision on the top surface of the artery about one third of the way down from the top of the isolated area. Insert the tip of the cannula into the artery with a of curved tip forceps. Once inserted, remove the straight fine tip forceps, occluding the blood flow and advance the cannula 2.5 centimeters into the artery using two pairs of forceps.

One to hold the cannula inside the artery to stop blood from leaking back and the other to insert the cannula. Then use a small artery clamp to hold the cannula inside the artery. Inject anticoagulant solution into the cannula, followed by drawing back a small amount of blood to confirm the patency of the tubing.

Then flush the cannula with anticoagulant solution and seal the end using the flame of a lighter. Finally, use the sutures to tie the cannula in place and remove the artery clamp. Then secure a third suture around the artery and cannula, shorten the length of the sutures around the artery and close the neck wound with more sutures following the recovery period, infused a formulation of interest into the animal via the duodenum cannula.

As illustrated in the graph after administration of the model, lipophilic drug halal fanene, the mean lymph flow rate for successfully cannulated rats was between 0.4 to 1.3 milliliters per hour. In some animals, the lymph flow rate was slightly lower during the first one to three hours following cannulation before increasing a commonly observed phenomenon following successful lymph cannulation in unsuccessfully cannulated rats. However, the flow rate remains substantially lower than that seen in the successful experiments throughout the collection period.

Similarly, in these representative experiments, the cumulative lymphatic transport of triglyceride and halal RIN was substantially lower in the unsuccessfully cannulated animals. Whereas in the successfully cannulated groups, the mean cumulative halalan transport was 15.1%of the dose and the cumulative triglyceride transport was 61 milligrams over 10 hours. In this representative lipid transport experiment, the proportion of the administered radio labeled exogenous lipid dose transported in the lymph was 54%These values were not significantly different than those observed previously.

For a similar formulation, the transport rate of both triglycerides and drugs into the lymph peaks, a few following lipid and drug administration, and then returns to baseline levels. As is typical and intestinal lymphatic lipid and drug transport experiments. The rate of drug transport in the lymph during each time period mirrors that scene for triglyceride transport as the drug is transported into the lymph.

In association with the triglyceride rich lipoproteins Once mastered, the cannulation can be completed within 30 minutes if no difficulties are encountered. Following this procedure, the components within collected lymph may be isolated and used for additional experiments such as for reinfusion to recipient animals to directly evaluate their function, metabolism, clearance, or disposition patterns. After watching this video, you should have a good understanding of the techniques to isolate and cannulate the mesenteric lymph duct, the carotid artery, and the duodenum.

Using these techniques, you should be able to assess the transport of both drugs, lipids, and other factors from the intestine through and into the mesenteric lymph in Viva.