Intraduodenal Cannulation in a Rat Model for Immunopathology Studies

Protocol

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

1. Preparations the Day Before the Surgical Procedure

1. Fast the rat the night before surgery if required. Ensure the rat has free access to water.
2. Prepare the solutions to be administered to the rat.
   1. Prepare a rehydration solution such as sterile saline or Ringers solution.
   2. Prepare anesthetic solutions as required. The anesthetic used in the experiments described here consisted of  "Cocktail 1" prepared by combining 1.9 ml of ketamine 100 mg/ml, 0.5 ml of Xylazine 100 mg/ml, 0.2 ml of acepromazine 10 mg/ml, and 2.5 ml of saline and "Cocktail 2" consisting of 1 ml of ketamine 100 mg/ml and 0.1 ml acepromazine 10 mg/ml. NOTE: Inhaled isoflurane or sevoflurane may be preferred as it can provide a surgical plane of anesthesia for a longer period of time.
3. Prepare an anti-coagulant solution containing 10 mg/ml ethylenediaminetetraacetic acid (EDTA) in sterile water or 10 IU/ml heparin in saline to flush the lymph cannula. Also, prepare an anti-coagulant solution containing 2.5 - 10 IU/ml heparin in saline to flush the carotid artery cannula.
4. Prepare the polyethylene cannula for insertion into the duodenum (0.96 mm O.D, 0.58 mm I.D.)
   1. Cut the cannula to the required length (typically 25 - 30 cm for the duodenal cannula) and place a bevel at the tip of the cannula using a sterile surgical blade (see Figure 1).   
      NOTE: This increases the ease of cannula insertion and reduces the incidence of cannula blockage post-insertion.
   2. Place a J-shaped anchor point in the intraduodenal cannula by looping a small portion of the cannula back on itself, heating it with a lighter or hot plate, and then cutting it to size (see Figure 1).
5. Attach the lymph cannula to a 25 G needle and syringe containing an anti-coagulant solution (prepared in step 1.3). Fill the lymph cannula with the anti-coagulant solution and leave the solution in the cannula overnight (O/N) to reduce the incidence of clot formation in the lymph cannula during lymph collection.
6. Prepare tubes for lymph and blood sample collection. 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 ethylenediaminetetraacetic acid (EDTA) in sterile water or 10 - 20 IU/ml heparin in the collected lymph or blood.

2. Preparations Immediately Prior to Commencing the Surgical Procedure

1. Check the cannulas by flushing them with sterile saline or anti-coagulant solution to ensure that they are patent.
2. Prepare the rat for the surgical procedure.
   1. Anesthetize the rat throughout the surgery and sample collection period.
      1. For example, initiate the anesthesia via subcutaneous injection of 1.5 ml/kg of Cocktail I (from step 1.2.2) into a skin fold at the back of the rat neck using a 1 ml syringe attached to a 25 G needle. Maintain the anesthesia via intraperitoneal injection of  0.44 ml/kg of Cocktail 2 (from step 1.2.2) approximately each hour as required, using a 1 ml syringe attached to a 25 G needle.
      2. Prior to commencing the surgery, ensure that the depth of anesthesia is sufficient by observing respiratory rate, whisker movement, muscle tone, and responses to stimuli such as pinching of the foot. Administer additional anesthesia as required.
   2. Shave the fur from the surgical regions, which include the right side of the abdomen, for duodenum cannulation.
   3. Clean the surgical regions aseptically using povidone-iodine solution or chlorhexidine solution and a 70% ethanol scrub. Repeat this 3 times for each region, finishing with a final cleansing with 70% ethanol.
3. Place the animal in dorsal recumbence on a clean sheet above a heated surgical pad (37 °C).

3. Cannulation of the Duodenum

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 2B).
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 2B).
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. Attach the duodenum cannula to a syringe (e.g., 10 ml) filled with the rehydration solution.
6. Identify the duodenum as the bright pink (with more blood vessels) section of the small intestine, which upon gentle downward pulling reveals the stomach.
7. Make a small puncture hole in the duodenum approximately 2 cm below the junction of the stomach and duodenum (the pylorus) using a sterile 23 G needle.
8. Insert the J-shaped hooked end of the duodenal cannula through the puncture hole. Secure in place with a drop of cyanoacrylate glue.
9. Commence hydration of the rat by attaching the cannula to the rehydration syringe loaded in an infusion pump. According to the literature, rehydration rates range from 0.5 - 3 ml/hr.
10. After completion of the lymph and duodenum cannulation, close the incision in the abdominal muscle wall with sutures. Then close the skin incision by applying a few drops of tissue adhesive (cyanoacrylate) along the side of the incision and pinching the skin on both sides of the incision together.

Results

Figure 1. Diagrammatic representation of the shape of the beveled tip of a carotid artery or lymph cannula, and the J-shaped beveled tip of a duodenal cannula.

Figure 2. Photographs of (A) the abdomen shaved in preparation to cannulate the lymp...

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
Betadine solution	Livingstone International	BU0510	Any brand can be used. http://www.livingstone.com.au/?PG=search_result&CAT=6&search
Ilium Ketamil (Ketamine 100 mg/ml)	PROVET VICTORIA	KETA I 1	http://www.provet.com.au/
Ilium Xylazil (Xylazine 100 mg/ml)	PROVET VICTORIA	TRO-3828	http://www.provet.com.au/
ACP 10 Injection (Acepromazine 10 mg/ml)	PROVET VICTORIA	VTG-DACP010020	http://www.provet.com.au/
Heparin (35000I.U. in 35 mL)	Sigma Pharmaceuticals	337220	http://sigmaco.com.au/
Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate	Sigma-Aldrich	E1644	Any brand can be used. Example here is disodium salt of EDTA from Sigma.
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 0.8x0.5 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 0.96x0.58 mm, 30 m
Ruler	Any		Any brand can be used
Markers	Any		Any brand can be used
Cigarette lighter	Any		Any brand can be used
Cyanoacrylate glue	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 light	Harvard Apparatus	72-0215 with 72-0267	Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_
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
Silk suture	Livingstone International	DTSK163019F4	Any brand can be used. Example here is * Email this item to my friend                   3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&CAT=6&search          =DTSK163019F4
Scalpel blades	Fine Science Tools (FST)	10020-00	Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191
Scalpel handle	Fine Science Tools (FST)	10004-13	Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&CategoryId=51
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
2 x Hemostats	Fine Science Tools (FST)	13010-12	Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&CategoryId=33
1 x Suture needle holder	Fine Science Tools (FST)	12001-13	Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&CategoryId=70