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), dexmedetomidine (15 &#181;g/kg). 
	Note: Add buprenorphine (0.005-0.02 mg/kg) in the pre-anesthetic drug cocktail for enhanced intra-operative pain control.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C) during surgical procedure.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Abdominal Surgery and Catheterization of the Intestinal Lymphatic Trunk
<ol>
	<li>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.</li>
	<li>Place a retractor at the cranial aspect of the surgical incision to keep the abdominal cavity open for the duration of the surgery.</li>
	<li>Moisten all tissues with warm (37 &#176;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.</li>
	<li>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 pancreas22,36,37. Identify the lymphatic vessel as a translucent structure juxtaposed to the right ventral segment of the portal vein36,37.</li>
	<li>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 &#34;tunnel&#34; opening underneath the vessel with fine blunt tipped forceps.</li>
	<li>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 &#39;single loose ligature tie&#39; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place a specialized jacket on the pig while still anesthetized to ease its placement and reduce the stress of the pig during recovery.</li>
</ol>
3. Post-surgical Recovery and Lymph Collection
<ol>
	<li>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.</li>
	<li>Monitor the pigs for post-surgical complications every 8-12 hr for a 7 day period.</li>
	<li>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.</li>
</ol>
4. Quantification of Lipoprotein ApoB48, Triglyceride, Cholesterol and Total Protein Collected from Lymph
<ol>
	<li>Centrifuge the lymph sample at 1,800 x g for 5 min at 4 &#176;C. Collect the supernatant and use it for the quantification of triglyceride, cholesterol and total protein.</li>
	<li>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.</li>
	<li>Use the undiluted supernatant to measure cholesterol levels, with a commercially available kit.</li>
	<li>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.</li>
</ol>
5. Quantification of Lipoprotein ApoB48, Collected from Lymph38
<ol>
	<li>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).</li>
	<li>Transfer the separated proteins to a polyvinylidene fluoride membrane (0.45 &#181;m) and incubate them with a goat polyclonal antibody to ApoB (1:4,000), then bind it with an anti-goat secondary antibody.</li>
	<li>Quantify the lipoprotein ApoB48 with chemiluminescence using a linear densitometric comparison with purified rodent ApoB48 protein standard.</li>
</ol>

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 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 &#39;towel sling&#39; 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 &#39;pulling-out of the catheter&#39; 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&#39;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 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><tbody><tr><td>Miller laryngoscope blade</td><td>Welch Allyn</td><td>68044</td><td>182 mm length</td></tr><tr><td>Surgivet advisor: Vital signs monitor</td><td>Surgivet</td><td>V9203</td><td></td></tr><tr><td>Rectal temperature probe</td><td>Surgivet</td><td>V3417</td><td></td></tr><tr><td>Mono-polar electrosurgery generator</td><td>Valley Lab</td><td></td><td></td></tr><tr><td>Metzenbaum scissors</td><td>Fine Science</td><td>14518-18</td><td></td></tr><tr><td>Tuffier retractor</td><td>Stevens</td><td>162-11-676</td><td></td></tr><tr><td>Mosquito forceps</td><td>Stevens</td><td>162-7-10</td><td></td></tr><tr><td>Kelly forceps-curved (14cm)</td><td>Stevens</td><td>162-7-38</td><td></td></tr><tr><td>Allis tissue forceps</td><td>Stevens</td><td>162-7-38</td><td></td></tr><tr><td>Forceps dressing-eye (10.2cm)</td><td>Stevens</td><td>162-18-780</td><td></td></tr><tr><td>Forceps dressing-Adison (12.1cm)</td><td>Stevens</td><td>162-17-2510</td><td></td></tr><tr><td>Needle Drivers</td><td>Stevens</td><td>162-V98-42</td><td></td></tr><tr><td>Iris scissors</td><td>Fine science</td><td>14058-11</td><td></td></tr><tr><td>Circulating water pump</td><td>Jorvet</td><td>J-783X</td><td></td></tr><tr><td>Maxitherm-Vinyl blanket</td><td>Jorvet</td><td>J-784C</td><td></td></tr><tr><td>Q tip applicators</td><td>Fisher Scientific</td><td>22-037-960</td><td></td></tr><tr><td>Catheterization&amp;nbsp; tubing (4.06 OD X 2.31 ID)</td><td>Braintree Scientific Inc.</td><td>MRE-160</td><td>Micro-Renethane implantation tubing</td></tr><tr><td>2-0 silk suture</td><td>Ethicon</td><td>LA556</td><td></td></tr><tr><td>2-0 polyglactin suture</td><td>Ethicon</td><td>J443H</td><td>2-0 vicryl</td></tr><tr><td>Large animal jacket</td><td>Lomir Biomedical Inc.</td><td>SSJ2YC</td><td></td></tr><tr><td>Polypropylene wash bottles</td><td>Fisher Scientific</td><td>03-409-22C</td><td>500 ml</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>D4333</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>60-00-4</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma Aldrich</td><td>A2411</td><td></td></tr><tr><td>Azaperone</td><td>Elanco Animal Health</td><td></td><td>Stresnil</td></tr><tr><td>Dexmedetomidine hydrochloride</td><td>Zoetis</td><td>6295</td><td>Dexdomitor</td></tr><tr><td>Isoflurane</td><td>Abbott Animal&amp;nbsp; Health</td><td>05260-5</td><td>IsoFlo</td></tr><tr><td>Ketamine hydrochloride</td><td>Zoetis</td><td>2626</td><td>Ketaset</td></tr><tr><td>Bupenorphine hydrochloride</td><td>Champion Alstoe Animal Health</td><td></td><td>DIN:02347510</td></tr><tr><td>6 mm Endotracheal tube</td><td>Jorvet</td><td>J-165d</td><td></td></tr><tr><td>10% Lidocaine spray</td><td>AstraZeneca</td><td></td><td>DIN:02003767</td></tr><tr><td>4 % Chlorhexidine surgical scrub</td><td>Partnar Animal Health</td><td>PCH-011</td><td>Diluted: 2.0% solution</td></tr><tr><td>3M Surgical steri- drape</td><td>3M Health Care</td><td>1040</td><td></td></tr><tr><td>SDS page gel</td><td>Invitrogen</td><td>EA0375BOX</td><td>3-8 % tris acetate</td></tr><tr><td>Polyvinylidene fluoride membrane</td><td>Millipore</td><td>IPVH00010</td><td>0.45 &amp;mu;m pore size</td></tr><tr><td>ApoB antibody&amp;nbsp;</td><td>EMD Millipore</td><td>AB742</td><td>1:4000 dilution</td></tr><tr><td>Donkey anti-goat IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>Sc-2304</td><td></td></tr><tr><td>ECL Prime Western Blotting Reagent</td><td>GE Healthcare LifeSciences</td><td>RPN2232&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Triglyceride Kit</td><td>Wako Pure Chemicals</td><td>998-40391/994-40491</td><td></td></tr><tr><td>Total Cholesterol Kit</td><td>Wako Pure Chemicals</td><td>439-17501</td><td></td></tr><tr><td>Total Protein</td><td>&amp;nbsp;Pierce&amp;nbsp;</td><td>23225</td><td>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 in-line with values of central lymph reported by other investigators following catheterization of intestinal lymphatic vessels (lymph flow 570 &#177; 158 to 979 &#177; 284 ml/24 hr25, 360 &#177; 120 mLl/24 hr to 1080 &#177; 720 ml/24 hr26; triglyceride 687 &#177; 110 mg/dL9; cholesterol 63.1 &#177; 5.6 mg/dL9); indicating proper placement of the catheter within the lymphatic vessel.
<img alt="Figure 2" src="/files/ftp_upload/53457/53457fig2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53457/53457fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53457/53457fig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53457/53457fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Lymph Characteristics&#160;</td>
		</tr>
		<tr>
			<td>ApoB48 (mg/ml)</td>
			<td>3022.4 &#177; 440.4</td>
		</tr>
		<tr>
			<td>Triglyceride (mg/dL)</td>
			<td>607 &#177; 203.6</td>
		</tr>
		<tr>
			<td>Cholesterol (mg/dL)</td>
			<td>58.5 &#177; 9.6</td>
		</tr>
		<tr>
			<td colspan="2">Results are expressed as mean &#177; SD (n=3)</td>
		</tr>
	</tbody>
</table>
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)

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

8.6K Views.  University of Alberta. 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.

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

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Immunology and Infection

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

Biology

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

Intubation, Central Venous Catheter, and Arterial Line Placement in Swine for Translational Research in Abdominal Transplantation Surgery

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries does not solely rely on the research team's surgical skills; perioperative care and management procedures, like intubation, central venous line, and arterial line placement, are necessary and of the utmost importance for favorable experiment results. As it is uncommon for research teams to have anesthesiologists or any other staff other than the surgical team, the surgical team involved in translational research must acquire and/or develop the skills to perform the perioperative care. The purpose of this paper is to show the techniques of intubation, central venous catheter, and arterial line placement used and perfected at the Toronto Organ Preservation Laboratory over the last 10 years, to be used as a reference for future researchers joining either this team or any other lab performing translational research protocols in swine and/or abdominal transplantation.

To obtain the best possible results, surgeons performing translational research experiments should be proficient at intubation and vascular line placement. This paper describes the techniques used by the Toronto Organ Preservation Laboratory to perform these procedures.

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries ...

Hickman Catheter for Long-Term Vascular Access in Swine

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood monitoring and reliable intravenous fluid and drug administration. Specifically, the tunneled multi-lumen Hickman catheter (HC) is commonly used in swine models due to its lower extrication and complication rates. Despite fewer complications relative to other CVCs, HC-related morbidity presents a significant challenge, as it can significantly delay or otherwise negatively impact ongoing studies. The proper insertion and maintenance of HCs is paramount in preventing these complications, but there is no consensus on best practices. The purpose of this protocol is to comprehensively describe an approach for the insertion and maintenance of a tunneled HC in swine that mitigates HC-related complications and morbidity. The use of these techniques in &gt;100 swine has resulted in complication-free patent lines up to 8 months and no catheter-related mortality or infection of the ventral surgical site. This protocol offers a method to optimize the lifespan of the HC and guidance for approaching issues during use.

Author Spotlight: Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model  

A reliable and reproducible approach for the insertion and maintenance of a tunneled Hickman catheter for long-term vascular access in swine is described. Placement of a central venous catheter allows for convenient daily sampling of whole blood from awake animals and intravenous administration of medication and fluids.

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood ...

Surgical Technique for Lumbar Spinal Catheter Insertion in Pigs Enabling Continuous Access to the Thecal Sac in a Terminal Setup

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine and human central nervous systems (CNS). However, accessing the cerebrospinal fluid (CSF) in larger pig breeds by conventional lumbar puncture techniques can be challenging due to an oblique orientation of the spinal spinous processes and a limited interlaminar space. Accordingly, an open surgical procedure for inserting a lumbar spinal catheter for continuous CSF sampling at the L4/L5 level in pigs is thoroughly described in this work. After positioning the pig and identifying the anatomical landmarks, a dorsal midline surgical incision is made to expose the spinous processes. By advancing the introducer needle, the spinal catheter is inserted inside the thecal sac of the spinal canal while leaving the bone structures of the spine intact. This method allows continuous infusion into or sampling from the porcine thecal sac with minimal bleeding or CSF leakage. The procedure is simple, time-efficient, and reproducible across different experimental setups, offering significant potential for various pre-clinical studies, including pharmacokinetic research, surgical training, and spinal cord injury models.

We present a technique for inserting a lumbar spinal catheter at the L4-L5 level in a 3-month-old Danish Landrace pig as part of a terminal research protocol, enabling continuous infusion or CSF sampling from the thecal sac.

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine ...

Neuroscience

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

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

Source: Trevaskis, N. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52389">The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport.</a> J. Vis. Exp. (2015).
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.

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

Immunology

Immunopathology

Assay and Analysis

Intraduodenal Cannulation in a Rat Model for Immunopathology Studies

Source: Trevaskis, N. L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52389">The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates intraduodenal cannulation in a rat model. After anesthetizing the rat, the duodenum is located and a small puncture is created. A J-shaped cannula with a beveled tip is carefully inserted and secured, and the incision is closed. The cannulated rat model enables intra-intestinal infusion for immunopathology studies.

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

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