Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

Overview

This video demonstrates a technique to cannulate the mesenteric lymph duct in rats. This method enables direct access to the entire volume of components present in the lymph fluid.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Cannulation of the Mesenteric Lymph Duct

1. Place the animal with its right side facing toward the operator. Perform the surgery with or without the assistance of a surgical microscope as required.
2. Open the top layer of the abdominal muscle wall with a straight 4 cm incision extending from the midline (xiphoid process) to the right flank approximately 2 cm below the ribcage (costal margin) using a sterile scalpel blade (see Figure 1B).
3. Open the remaining layers of the abdominal muscle wall from 4 - 5 mm lateral to the midline to the right flank with a small pair of surgical scissors (see Figure 1B).
4. Retract the small intestine under the left abdominal muscle wall and keep it in place using 2 - 3 pieces of sterile gauze saturated with normal saline.
5. Bridge the rat over a 10 ml plastic syringe placed horizontally under the rat's back at the level of the right kidney to ease visualization of the mesenteric lymph duct.
6. Locate the superior mesenteric lymph duct: a vessel approximately 0.5 - 1 mm in diameter that is perpendicular to the right kidney and immediately rostral and parallel to the mesenteric artery (a pulsating dark red blood vessel; see Figure 1C).
   NOTE: In non-fasted or oil-pre-dosed rats the vessel is white, opaque, and easier to visualize than in fasted rats in which it is quite translucent.
7. Inspect the area immediately caudal to the large superior mesenteric lymph duct and the mesenteric artery to determine if a second, smaller accessory lymph duct is present. If present, block the flow of lymph through the duct by severing the duct and occluding with superglue, or tying a suture around the duct, to ensure that the entire volume of lymph flowing from the intestine is collected from the superior mesenteric lymph duct.
8. Pass a pair of straight forceps through the peri-renal fat bed at the lower margin of the right kidney, and through the connective tissue layers immediately below the vena cava, in a direction parallel with the superior mesenteric lymph duct.
9. Using the tip of the forceps take hold of the lymph cannula and pull it through the peri-renal 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.
10. Isolate the mesenteric lymph duct from overlying layers of connective and fat tissue by blunt dissection. Take care not to pierce or damage the lymph duct.
11. After isolating the lymph duct, make a small hole in the lymph duct with either micro-scissors, the sharp tip of the jeweler's forceps, or a 25 G needle. Take care not to completely sever the vessel.
12. Ensure that the lymph cannula is completely filled with an anti-coagulant solution (using a syringe and 25 G needle) with no air gaps. Insert the lymph cannula approximately 2 - 4 mm inside the mesenteric lymph duct via the small hole with the aid of a small pair of forceps.
13. Observe the lymph cannula for a couple of minutes. Observe a gradual flow of intestinal lymph from the free-collecting end of the cannula if the cannulation is successful. If the cannulation is not successful, repeat steps 1.8 - 1.12.
14. If the cannulation is successful, secure the cannula by placing a small drop of veterinary adhesive over the entrance hole into the lymph duct. Take care not to occlude the vessel via the application of excess veterinary adhesive.
15. Whilst waiting for the veterinary adhesive to set, observe the flow of lymph for several minutes to ensure that the cannulation procedure was successful. Once certain of the success of the cannulation, remove the gauze pieces that were placed in the abdominal cavity carefully and place the small intestine in its original position.
16. Pre-weigh lymph collection tubes, label tubes for lymph and blood sample collection and add anti-coagulant solution. Add sufficient anti-coagulant solution to achieve a final concentration of 1 mg/ml EDTA in sterile water or 10 - 20 IU/ml heparin in the collected lymph or blood.
17. Place a lymph collection tube containing an anti-coagulant (step 1.16) at the end of the cannula to collect the free-flowing lymph.
18. Next, cannulate the duodenum for infusion of rehydration solutions and/or formulations.

Representative Results

Figure 1. Photographs of (A) the abdomen shaved in preparation to cannulate the lymph duct, (B) the abdominal muscle wall opened with a straight 4 cm incision extending from the midline to the right flank to access the lymph duct, and (C) the superior mesenteric lymph duct (in yellow circle) perpendicular to the right kidney.

Materials

Name	Company	Catalog Number	Comments
Sterile saline	Baxter healthcare	AHB 1307	Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50
70 % ethanol in water	Any		Any brand can be used
Chlorhexidine gluconate solution (Microshield 4)	Livingstone International	JJ60243L	Any brand can be used. http:// www.livingstone.com.au/? PG=search_result&CAT=6&search =JJ60243L
Betadine solution	Livingstone International	BU0510	Any brand can be used. http:// www.livingstone.com.au/? PG=search_result&CAT=6&search =BU0510
Ilium Ketamil (Ketamine 100 mg/ ml)	PROVET VICTORIA	KETA I 1	http://www.provet.vom.au
Heparin (35000I.U. in 35 mL	SigmaPharmaceuticals	337220	http://www.sigmaco.com.au
Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm	Microtube extensions	PE8050	Any brand can be used. Example here is PE tubing 0.96 x0.58 mm, 30 m
Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm	Microtube extensions	PE9658	Any brand can be used. Example here is PE tubing 0.8 x0.5 mm, 30 m
Markers	Any		Any brand can be used
Veterinary adhesive	Any		Any brand can be used
23 gauge needles	Livingstone International	DN23GX0.75LV	Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http:// www.livingstone.com.au/? PG=search_result&CAT= 6&search=DN23GX0.75LV
25 gauge needles	Livingstone International	DN25GX1.0LV	Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http:// www.livingstone.com.au/? PG=search_result&CAT=6&search= DN25GX1.0LV
1 ml syringe	Livingstone International	T3SS01TA	Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http:// www.livingstone.com.au/? PG=search_result&CAT=6&search =T3SS01TA
10 ml syringe	Livingstone International	T3SS10SA	Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http:// www.livingstone.com.au/? PG=search_result&CAT=6&search =T3SS10SA
Gauze swabs	Livingstone International	GSC075	Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http:// www.livingstone.com.au/? PG=search_result&CAT=6&search =GSC075
Heating pad	Ratek	WT1	Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.
Surgical microscope	Zeiss	495005-0014-000	Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https:// www.micro-shop.zeiss.com/? l=en&p=us&f=e&i=10143
1 x Small surgical scissors	Fine Science Tools (FST)	14060-09	Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/ Special-Pages/Products.aspx? ProductId=40&CategoryId=17
2 x Forceps with serrated curved tip	Fine Science Tools (FST)	11001-13	Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/ Special-Pages/Products.aspx? ProductId=405&CategoryId=32