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

Summary

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.

Abstract

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.

Introduction

The lymphatic system is an understudied area of physiology. Preclinical models of lymphatic catheterization occur in different animal species^1-8 and are used by pharmaceutical industries and research institutions to investigate mechanisms involved in lipid^8-12 and drug metabolism^13-15, cancer metastasis¹⁶ with experimental treatment¹⁷, and immune function^18-26. 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 people^27-31.

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 approach^32-34, and a ventral midline or paramedian approach^22,35. 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.

Protocol

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

1. 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), dexmedetomidine (15 µg/kg).
   Note: Add buprenorphine (0.005-0.02 mg/kg) in the pre-anesthetic drug cocktail for enhanced intra-operative pain control.
2. Anesthetize the pigs with inhaled isoflurane inhalation gas (4-5% isoflurane at 500 ml-1,000 ml/min O₂) using a face mask. Visualize the vocal cords using a veterinary laryngeal scope (17-25 cm long straight blade), and apply topical 10% lidocaine spay to the vocal cords. Allow the lidocaine spray to contact the vocal cords 30-60 sec prior to intubation to reduce the possibility of vocal cord spasm and airway obstruction.
3. Intubate the pigs by passing a cuffed endotracheal tube (5.0-7.0 mm Internal Diameter (ID)) between the vocal cords and maintain the anesthesia with isoflurane gas (0.5-2.0% isoflurane at 1,000-2,000 ml/min O₂) using a closed circuit rebreathing anesthetic system throughout the surgery. Assess the level of anesthesia by jaw tone, and both pedal and palpebral reflex responses. Expired anesthetic gas is scavenged and vented outside the surgical suite.
4. Clean the exterior surface of the ear with 2.0% chlorhexidine surgical scrub solution followed by a 70% isopropyl alcohol rinse. With a 20 G intravenous catheter, catheterize an ear vein to provide intravenous fluids (Lactated Ringer's Solution; 5-10 ml/kg/hr) during the surgery. Secure a pulse oximeter to the mucosal surface of the tongue with medical tape to monitor heart rate and the saturation of peripheral blood oxygenation (SpO₂).
5. Place the anesthetized pig in a dorsal recumbent position and shave the ventral abdomen from the mid thorax caudally to the ventral aspect of the pubis. Clean this area with two alternating 2.0 % chlorhexidine surgical scrubs and sterile water washes.
6. Transfer the anaesthetized pig to the surgical suite and apply the final surgical scrub of 70% isopropyl alcohol rinse, allow it to dry, and then drape the animal.
7. Insert a rectal temperature probe approximately 2-4 cm into the rectum to monitor body temperature. Place the pigs on a water recirculating heating pad to maintain normal body temperature (38-40 °C) during surgical procedure.
8. Drape the pig with four towels drapes placed in an overlying quadrant pattern around the abdomen. Place the first drape across xiphisternum, the second drape along lateral aspect of the abdomen approximately 5 cm lateral to the abdominal midline. Place the third drape across the ileal crest of the pelvis and the fourth drape, like the second drape (although on the opposite side), is placed along the lateral aspect of the abdomen approximately 5 cm lateral to the abdominal midline.
9. Place a large table drape, with a slit-opening allowing access to the surgical site, over the underlying towel drapes and cover the pig and entire surgical table. The final drape is a disposable steri-drape placed over the large table drape.

2. Abdominal Surgery and Catheterization of the Intestinal Lymphatic Trunk

1. Make a 20 cm skin incision with a scalpel blade to expose the underlying abdominal muscles. Incise the abdominal muscle layers with mono-polar electrocautery (20 Watts setting) to expose the parietal peritoneum. Open a 20 cm linear segment of parietal peritoneum with Metzenbaum scissors to access the abdominal viscera and lymphatic vessel.
2. Place a retractor at the cranial aspect of the surgical incision to keep the abdominal cavity open for the duration of the surgery.
3. Moisten all tissues with warm (37 °C) sterile saline for the entire surgical procedure. Gently lift a large segment of intestine including the colon, cecum, ileum and jejunum from the abdominal cavity and exteriorize it to the left flank of the pig to access the upper abdomen, liver and lymphatic vessel. Secure the exteriorized intestine in position with additional towel drapes to form a sling to gently support the intestine.
4. Locate the lymphatic vessel, it lays approximately 4 cm cranial-medial of the right renal vein, 6 cm caudal-medial- ventral of the caval foremen and underneath the visceral aspect of the right liver lobe near the pancreas^22,36,37. Identify the lymphatic vessel as a translucent structure juxtaposed to the right ventral segment of the portal vein^36,37.
5. Separate the lymphatic vessel from the surrounding fascia by gently teasing away the attached tissue with Q-tip applicators. Once the lateral aspects of vessel are separated from the surrounding tissue, create a "tunnel" opening underneath the vessel with fine blunt tipped forceps.
6. Pass three 2-0 silk sutures underneath the lymphatic vessel with fine forceps. Ligate the most caudal suture first to occlude, dilate and fill the vessel with lymph. Purposefully leave the ends of this suture relatively long (4 cm) to secure the catheter to the lymphatic vessel. Place two other sutures separated 1.0 cm from each other and are approximately 1.0-1.5 cm cranial to the secured caudal suture. Leave these two sutures with a 'single loose ligature tie' to allow for faster securing of the catheter into the vessel.
   Note: The segment of the lymphatic vessel located between the most caudal ligating suture and middle suture (of the two cranial sutures) is the site for catheterization. Regarding suture material, 2-0 polyglactin suture can substitute for 2-0 silk sutures if required.
7. Cut a small hole into the vessel with iris scissors and dilate the vessel with fine blunt forceps. Insert approximately 1.0-1.5 cm of specialized catheter tubing (4.06 Outer Diameter (OD) X 2.31 mm ID) with a beveled end into the vessel and tie the two cranial sutures to secure the catheter in place. Use the long suture ends of the caudal suture to secure the catheter to the vessel.
8. Wash the exteriorized intestine with copious amounts of warm saline and gently return it to the abdominal cavity ensuring correct anatomical positioning of the gut.
9. Exteriorize the catheter at the left mid flank (5-10 cm ventrally from the paralumbar fossa). Make a skin incision with a scalpel and pass a trocar from the abdominal cavity to the skin surface to create an opening for the exteriorization of the catheter. Use a large Kelly forceps to exteriorize the catheter from the abdominal cavity through the trocar opening.
10. Close the parietal peritoneum with a simple continuous suture pattern of 2-0 polyglactin suture with a round (tapered) needle. Close the abdominal muscle layers with a simple interrupted suture pattern with a 2-0 polyglactin suture on a round needle.
11. Close the skin in a subcuticular pattern with 2-0 polyglactin suture on a cutting needle. Secure the exteriorized catheter to the skin with a purse-string suture pattern and a 2-0 nylon suture on a cutting needle.
12. Place a specialized jacket on the pig while still anesthetized to ease its placement and reduce the stress of the pig during recovery.

3. Post-surgical Recovery and Lymph Collection

1. Approximately 10 min prior to the discontinuation of inhalation anesthesia, administer bupenorphine (0.1 mg/kg) intramuscularly to provide immediate post-surgery analgesia. Continue bupenorphine (0.1 mg/kg) every 12 hr for 24-48 hr to maintain post-surgery analgesia.
2. Monitor the pigs for post-surgical complications every 8-12 hr for a 7 day period.
3. Collect the lymph in 500 ml polypropylene wash bottles, coated with ethylenediamine tetraacetic acid (EDTA), and supplemented with antibiotics; penicillin (6,000 IU), streptomycin (6 mg) and amphotericin B (3 mg) every 12 hr for a 7 day period.

4. Quantification of Lipoprotein ApoB48, Triglyceride, Cholesterol and Total Protein Collected from Lymph

1. Centrifuge the lymph sample at 1,800 x g for 5 min at 4 °C. Collect the supernatant and use it for the quantification of triglyceride, cholesterol and total protein.
2. Divide the supernatant into three samples: an undiluted sample, a sample diluted 1:20 distilled water and a final sample diluted 1:100 with distilled water.
3. Use the undiluted supernatant to measure cholesterol levels, with a commercially available kit.
4. Use the 1:20 and 1:100 diluted samples to measure triglyceride and total protein levels with a commercially available kit and the bicinchoninic acid-total protein assay respectively.

5. Quantification of Lipoprotein ApoB48, Collected from Lymph³⁸

1. Determine the concentration of lipoprotein ApoB48 with an adapted immune Western Blotting method³⁸. Separate total lymph with 3-8% tris-acetate- sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2. Transfer the separated proteins to a polyvinylidene fluoride membrane (0.45 µm) and incubate them with a goat polyclonal antibody to ApoB (1:4,000), then bind it with an anti-goat secondary antibody.
3. Quantify the lipoprotein ApoB48 with chemiluminescence using a linear densitometric comparison with purified rodent ApoB48 protein standard.

Results

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 ...

Discussion

Collecting intestinal lymph is an excellent method to investigate mechanisms involved in lipid^8-12 and drug^13-15 metabolism, cancer metastasis^16,17, cell trafficking and immune function^18-26, 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...

Materials

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