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 (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.
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.
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
2. Abdominal Surgery and Catheterization of the Intestinal Lymphatic Trunk
3. Post-surgical Recovery and Lymph Collection
4. Quantification of Lipoprotein ApoB48, Triglyceride, Cholesterol and Total Protein Collected from Lymph
5. Quantification of Lipoprotein ApoB48, Collected from Lymph38
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 in-line with values of central lymph reported by other investigators following catheterization of intestinal lymphatic vessels (lymph flow 570 ± 158 to 979 ± 284 ml/24 hr25, 360 ± 120 mLl/24 hr to 1080 ± 720 ml/24 hr26; triglyceride 687 ± 110 mg/dL9; cholesterol 63.1 ± 5.6 mg/dL9); indicating proper placement of the catheter within the lymphatic vessel.
Figure 2. A Fast Protein Liquid Chromatography (FPLC) profile demonstrating the purity of the chylomicron preparation in neonatal pig intestinal lymph. Lymph collected from three pigs is separated using a 1.006 g/ml density gradient ultracentrifugation protocol. The profile shows a single peak for (A) cholesterol and (B) triglyceride in the lymph sample. Please click here to view a larger version of this figure.
Figure 1. A representative Western blot analysis demonstrating the contribution of ApoB48 and ApoB100 lipoproteins in neonatal pig intestinal lymph. Lymph collected from three pigs is separated using a 1.006 g/ml density gradient ultracentrifugation protocol. The figure demonstrates a small amount of contamination with approximately 15% of plasma derived ApoB100. Please click here to view a larger version of this figure.
Lymph Characteristics | |
ApoB48 (mg/ml) | 3022.4 ± 440.4 |
Triglyceride (mg/dL) | 607 ± 203.6 |
Cholesterol (mg/dL) | 58.5 ± 9.6 |
Results are expressed as mean ± SD (n=3) |
Table 1. Components of intestinal lymph collected from neonatal pigs 7 days post-surgery. Results are repeated measures of lymph and expressed as mean SD (n=3)
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 populations following challenges with immune modulating agents18-22,25,26. Similarly, collection of central lymph has been helpful in delineating processes involved in lipoprotein metabolism8-12. The utility of lymph collection is dependent on the success of the catheterization procedure and the patency of the catheter within the lymphatic vessel.
Catheterization of the major lymphatic vessels involved in intestinal lymph drainage in large animals generally uses one of three surgical approaches. The first technique uses a cranial shoulder approach to enter the thoracic duct overlying the common jugular vein (cow thoracic duct)5. The second uses an upper flank approach over the mid-lateral aspect of the right kidney32,33. The third technique as described in this video employs either a ventral midline or paramedian22,35 laparotomy to expose the intestinal lymphatic trunk near the pancreas and juxtaposed to the right ventral aspect of the portal vein22,36,37. Although both the upper flank and ventral midline surgical techniques enable catheterization of the intestinal lymphatic trunk, we believe the ventral midline approach allows for better exposure of the vessel with adequate space within the upper abdomen to handle and manipulate the vessel.
Importantly, as in any experimental surgery there are steps that are particularly crucial for the successful outcome of the experimental procedure. There are four key areas that require careful attention using the ventral midline approach to access the intestinal lymphatic vessel. First, excellent blood supply to the exteriorized intestine is required, as prolonged compromised circulation to the intestine induces ischemic intestinal mucosal necrosis and surgical failure. In this procedure a 'towel sling' reduces the tension on the mesenteric blood vessels by supporting the weight of the intestines ensuring excellent tissue perfusion. Second, careful manipulations of the vessel including: the release of the vessel from surrounding fascia and adipose tissue, inserting and firmly securing the catheter within the vessel for long-term lymph collection is critical for successful catheterization. Indeed, it is suggested that the structural integrity of the vessels in neonatal pigs is not as robust as in mature animals and as such can easily tear39,40. Using Q-tip applicators to slowly and gently separate fascial layers and fine blunt-ended instruments to pass the suture underneath the vessel greatly reduces the inadvertent puncture or tearing of the lymphatic vessel. Rough handling of the vessel often creates a friable vessel with weak structure making catheterization very challenging. Third, a common cause of catheterization failure is the unintentional 'pulling-out of the catheter' from the lymphatic vessel. Securing the catheter directly to the vessel reduces the incidence of catheter loss. This is accomplished by tying the catheter directly to the vessel with the caudal suture employed to dilate the vessel. Finally, it is important to ensure good lymph flow is present following catheterization and prior to abdomen closure. Indeed, if lymph flow is sluggish during the procedure, over time this induces clot formation within the catheter and markedly reduces the lymph collection period. In the investigator's experience, with long term lymph collection of non-intestinal lymph in sheep41, it is very difficult to remove the clot from the catheter as the clot develops within the catheter and is deep-seated within the lymphatic vessel. Consequently, this will greatly reduce (approximately 40%) the numbers of animals successfully catheterized for long-term experimentation (unpublished data). Therefore, if lymph flow is slowed or has discontinued during the procedure, the lymphatic catheter should be reinserted cranially 0.5-1 cm from the initial catheterization site.
All models used to investigate scientific questions have inherant limitations and catheterization of the intestinal lymphatic trunk is no exception. The most pronounced limitation for the procedure is the inability to effectively isolate, catheterize the lymphatic vessel, and stabilize the catheter within the vessel. As such, this procedure requires people with excellent technical and surgical skills. Another significant limitation is that prolonged surgical times during major abdominal surgery increases the risk and post-surgical morbidity and mortality. Notably, however, the pigs in our experiment recovered quickly from surgery and there were no complications such as infections, intestinal ileus, excessive tissue injury, or inappetence following the procedure. Most certainly, pigs were observed to be bright, responsive, ambulating and eating normally 24 hr post-surgery and this continued over the 7 day lymph collection period. Notably, although serum chemistries or complete blood counts were not assessed in these pigs following the 7 day experiment, previous work by the investigators in long-term lymph collection in sheep41 demonstrated that catheterized sheep were never leukopenic, lymphopenic, hypoproteinemic with electrolyte imbalances or clinically portraying poor health (unpublished data). Importantly and as stated above, this observation correlates to catheterized pigs also displaying clinical characteristics of good health.
Interestingly, investigators noted variations in the volume of lymph flow. The flow was greatest during the day when pigs were feeding and active with approximately 70% of the daily collected lymph occurring at this time. The remaining 30% of lymph flow happens in the evening during rest and sleep.
Further characterization of lymph samples are provided in Figures 1, 2 and Table 1. Figure 1 demonstrates a small amount of contamination with approximately 15% of plasma derived ApoB100. The presence of plasma lipoprotein within intestinal lymph is an observation that often occurs in pigs42. In suckling pigs, the majority of the lipoprotein is produced by the liver and released in plasma. From the plasma, ApoB100 likely seeps into collecting lacteals42 and then through the lymphatic vascular network drains into the intestinal lymphatic trunk. Various methods of chromatography are used to assess cholesterol, triglycerides and other phospholipid contents in collected lymph samples42-45. The single peak in Figure 2 clearly demonstrates cholesterol and triglycerides are present in the chylomicron fraction. This method is a convenient and accurate technique of profiling lymph lipoproteins and should be considered when assessing the purity of chylomicron samples from intestinal lymph45. Finally, Table 1 provides the biochemical components of intestinal lymph collected from neonatal pigs 7 days post-surgery.
In conclusion, long-term catheterization of the intestinal lymph trunk is successful in young pigs. With this technique, lymphatic catheterization can be used to collect and investigate components of lipid metabolism. The purity of lymph, amount of lymph produced and the quantities of constituents of lipid metabolism are similar to amounts present in pig central lymph in other experiments9,25,26,42. The results indicate that employing a ventral midline surgical approach with careful tissue dissection and vessel handling to catheterize the intestinal lymphatic trunk in young pigs is an excellent method to collect large quantities of central lymph.
The authors have nothing to disclose.
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.
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 |