The work was supported in part by funding from Alberta Livestock and Meat Agency and Natural Science and Research Council Discovery grant to S. D. Proctor.

All procedures on experimental animals described in both the video and manuscript were approved by the Institutional Animal Care and Use Committee and followed the guidelines set by the Canadian Council of Animal Care.
1. Surgical Anesthesia and Surgical Preparation of the Neonatal Pigs
<ol>
	<li>In a separated anteroom, premedicate 25 kg pigs near the base of the neck with an intramuscular sedative-anesthetic drug cocktail containing: azaperone (0.3 mg/kg), ketamine hydrochloride (10 mg/kg)...

The authors have nothing to disclose.

Collecting intestinal lymph is an excellent method to investigate mechanisms involved in lipid8-12 and drug13-15 metabolism, cancer metastasis16,17, cell trafficking and immune function18-26, in various experimental animal models. Indeed, the ability to harvest large quantities of either peripheral (afferent) and central (efferent and large trunk vessels) lymph over an extended period has been particularly important for understanding temporal changes that occur in cell populati...

The lymphatic system is an understudied area of physiology. Preclinical models of lymphatic catheterization occur in different animal species1-8 and are used by pharmaceutical industries and research institutions to investigate mechanisms involved in lipid8-12 and drug metabolism13-15, cancer metastasis16 with experimental treatment17, and immune function18-26. This study explores the use of intestinal lymph trunk catheterization in a domestic pig model to measure components of lipoprotein metabolism. Lipoprotein metabolism is involved in the production and secretion of chylomicrons, as well as changes in associated lipids and total protein. These are important considerations as there are major differences in lipid metabolism between commonly used rodent models and humans and as such, employing swine models to collect intestinal lymph could provide more comparable information for studying lipid metabolism in people27-31.
Several surgical techniques are used to collect the intestinal lymph in large animal species: a cranial shoulder approach (i.e., thoracic duct catheterization)5, a lateral upper flank approach32-34, and a ventral midline or paramedian approach22,35. This video describes in detail the surgical procedure in swine using a ventral midline surgical approach for the catheterization of the intestinal lymph trunk. Careful surgical technique permits this method of lymphatic catheterization to collect large quantities of lymph and its constituents over extended periods of time.
This technique opens a myriad of applications to many disciplines examining various physiologic functions. Applications could include, but are not limited to, whole body lipoprotein and lipid metabolism, immunosurveillance, tumor genesis and metastasis, intestinal function, and the development and progression of intestinal inflammatory disease.

<table border="0" cellpadding="0" cellspacing="0" width="567">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Miller laryngoscope blade</td>
			<td style="width:71px;">Welch Allyn</td>
			<td style="width:119px;">68044</td>
			<td style="width:167px;">182 mm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Surgivet advisor: Vital signs monitor</td>
			<td style="width:71px;">Surgivet</td>
			<td style="width:119px;">V9203</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Rectal temperature probe</td>
			<td style="width:71px;">Surgivet</td>
			<td style="width:119px;">V3417</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Mono-polar electrosurgery generator</td>
			<td style="width:71px;">Valley Lab</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Metzenbaum scissors</td>
			<td style="width:71px;">Fine Science</td>
			<td style="width:119px;">14518-18</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Tuffier retractor</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-11-676</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Mosquito forceps</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-7-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Kelly forceps-curved (14cm)</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-7-38</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Allis tissue forceps</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-7-38</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Forceps dressing-eye (10.2cm)</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-18-780</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Forceps dressing-Adison (12.1cm)</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-17-2510</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Needle Drivers</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-V98-42</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Iris scissors</td>
			<td style="width:71px;">Fine science</td>
			<td style="width:119px;">14058-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Circulating water pump</td>
			<td style="width:71px;">Jorvet</td>
			<td style="width:119px;">J-783X</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Maxitherm-Vinyl blanket</td>
			<td style="width:71px;">Jorvet</td>
			<td style="width:119px;">J-784C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Q tip applicators</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-037-960</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Catheterization&#160; tubing (4.06 OD X 2.31 ID)</td>
			<td style="width:71px;">Braintree Scientific Inc.</td>
			<td style="width:119px;">MRE-160</td>
			<td rowspan="2" style="width:167px;">Micro-Renethane implantation tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">2-0 silk suture</td>
			<td style="width:71px;">Ethicon</td>
			<td style="width:119px;">LA556</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">2-0 polyglactin suture</td>
			<td style="width:71px;">Ethicon</td>
			<td style="width:119px;">J443H</td>
			<td style="width:167px;">2-0 vicryl</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Large animal jacket</td>
			<td style="width:71px;">Lomir Biomedical Inc.</td>
			<td style="width:119px;">SSJ2YC</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Polypropylene wash bottles</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">03-409-22C</td>
			<td style="width:167px;">500 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Penicillin-Streptomycin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">D4333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">EDTA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">60-00-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Amphotericin B</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">A2411</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Azaperone</td>
			<td style="width:71px;">Elanco Animal Health</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Stresnil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Dexmedetomidine hydrochloride</td>
			<td style="width:71px;">Zoetis</td>
			<td style="width:119px;">6295</td>
			<td style="width:167px;">Dexdomitor</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Isoflurane</td>
			<td style="width:71px;">Abbott Animal&#160; Health</td>
			<td style="width:119px;">05260-5</td>
			<td style="width:167px;">IsoFlo</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Ketamine hydrochloride</td>
			<td style="width:71px;">Zoetis</td>
			<td style="width:119px;">2626</td>
			<td style="width:167px;">Ketaset</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:211px;">Bupenorphine hydrochloride</td>
			<td style="width:71px;">Champion Alstoe Animal Health</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">DIN:02347510</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">6 mm Endotracheal tube</td>
			<td style="width:71px;">Jorvet</td>
			<td style="width:119px;">J-165d</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">10% Lidocaine spray</td>
			<td style="width:71px;">AstraZeneca</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">DIN:02003767</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">4 % Chlorhexidine surgical scrub</td>
			<td style="width:71px;">Partnar Animal Health</td>
			<td style="width:119px;">PCH-011</td>
			<td style="width:167px;">Diluted: 2.0% solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">3M Surgical steri- drape</td>
			<td style="width:71px;">3M Health Care</td>
			<td style="width:119px;">1040</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">SDS page gel</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">EA0375BOX</td>
			<td style="width:167px;">3-8 % tris acetate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Polyvinylidene fluoride membrane</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">IPVH00010</td>
			<td style="width:167px;">0.45 &#956;m pore size</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">ApoB antibody&#160;</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">AB742</td>
			<td style="width:167px;">1:4000 dilution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Donkey anti-goat IgG-HRP</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">Sc-2304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:211px;">ECL Prime Western Blotting Reagent</td>
			<td style="width:71px;">GE Healthcare LifeSciences</td>
			<td style="width:119px;">RPN2232&#160;&#160;&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Triglyceride Kit</td>
			<td style="width:71px;">Wako Pure Chemicals</td>
			<td style="width:119px;">998-40391/994-40491</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Total Cholesterol Kit</td>
			<td style="width:71px;">Wako Pure Chemicals</td>
			<td style="width:119px;">439-17501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Total Protein</td>
			<td style="width:71px;">&#160;Pierce&#160;</td>
			<td style="width:119px;">23225</td>
			<td style="width:167px;">Bicinchoninic Acid Assay</td>
		</tr>
	</tbody>
</table>

long-term catheterization of the intestinal lymph trunk and collection of lymph in neonatal pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

Lymphatic catheterization of the intestinal lymphatic trunk of neonatal pigs allows collection of approximately 1 L/24 hr of central lymph over a 7 day period. The lymph collected in this experiments contained components of lipid metabolism, namely total lymph protein, ApoB48 lipoprotein, triglycerides, total protein, and cholesterol. Table 1 highlights representative amounts of these lipid components from pooled lymph samples of three pigs. Notably, lymph flow and lipid constituents are ...

Watch this Scientific Journal Video about Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs at JoVE.com

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Lymphatic catheterization is an important technique used to investigate mechanisms involved in lipid and drug metabolism, cancer metastasis, and immune function and collecting large quantities of lymph from various animal models enables researchers to study these processes. The video demonstrates in detail the surgical procedure employed to catheterize the intestinal lymphatic trunk in neonatal pigs using a ventral mid-line approach. All procedures were approved by the University of Alberta Institutional Animal Care and Use Committee.To access the lymphatic vessel the abdominal cavity was opened. Electrocautery on the cut setting was used to open the muscle layer. A retractor was placed to hold the abdominal wall open for the duration of the procedure.Key abdominal organs are identified to aid in localizing the lymphatic vessel. For improved access to the lymphatic vessel segments of the small intestine and colon are exteriorized to the left side of the body. The lymphatic vessel is landmarked by identifying the liver and the right ventral segment of the portal vein.In neonatal pigs the lymphatic vessels are delicate and easily torn, so meticulous dissection with swabs and blunt instruments is required. Here is the intestinal lymphatic vessel. Now the suture is being passed underneath the lymphatic vessel with blunt forceps.Now there are three 2-0 silk sutures passed underneath the lymphatic vessel. The caudal most suture is used to dilate the vessel and secure the catheter to the vessel. The cranial sutures are used to secure the catheter within the vessel.A small incision in the lymphatic vessel allows blunt forceps to dilate the vessel. The catheter is inserted into the vessel. This must be done gently to ensure the lymphatic vessel does not tear.The catheter needs to be advanced beyond the cranial most suture to ensure it is well seated. The three 2-0 silk sutures secure the catheter into the lymphatic vessel. Importantly the two cranial sutures are first used to secure the catheter into the vessel and the caudal most suture is then used to tether the catheter, adding stability.It is important to ensure there is good lymph flow prior to closing the abdominal cavity. The catheter is exteriorized on the left mid-flank of the pig. Large forceps are used to pull the catheter from the abdominal cavity to the external surface of the pig's flank.The muscle layers are closed with 2-0 vicryl suture on a tapered needle in a simple interrupted suture pattern. The skin is closed with 2-0 vicryl suture on a cutting needle in a running subcuticular suture pattern. With the exit incision the catheter passes through the skin and is closed with 2-0 nylon suture on a cutting needle in a purse-string suture pattern.This closure helps prevent ascending infections tracking inwards along the catheter. The jacket holding the lymph collection bottles is placed on the pig while the pig is still under anesthesia. The catheter is inserted into the collection bottle and good lymph flow is assured prior to waking the pig.The pigs recover well from the procedure and excellent lymph flow is maintained throughout the experiment. The table displays the components of lymph collected from the lymphatic vessel. These components include Apolipoprotein B48, Triglycerides, and Cholesterol.The figure of the Western Blot shows the presence of both Apolipoprotein B48 and small amounts of plasma-derived Apolipoprotein B100 within the collected lymph sample. Although lymphatic catheterization in young pigs can be technically challenging, as the lymphatic vessels are both delicate and friable, this video demonstrates that using proper and careful surgical technique in young pigs can provide researchers with an excellent model for collecting intestinal lymph.

long-term-catheterization-intestinal-lymph-trunk-collection-lymph

Research

JoVE Journal

Medicine

8.5K 조회수.  University of Alberta. Lymphatic catheterization is an important technique used to investigate mechanisms involved in lipid and drug metabolism, cancer metastasis, and immune function and collecting large quantities of lymph from various animal models enables researchers to study these processes. The video demonstrates in detail the surgical procedure employed to catheterize the intestinal lymphatic trunk in neonatal pigs using a ventral mid-line approach. All procedures were approved by the University of Alberta I...