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 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>
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			<td height="40" style="height:40px;width:211px;">Surgivet advisor: Vital signs monitor</td>
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			<td height="20" style="height:20px;width:211px;">Rectal temperature probe</td>
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			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Mono-polar electrosurgery generator</td>
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			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Metzenbaum scissors</td>
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			<td style="width:119px;">14518-18</td>
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		</tr>
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			<td height="20" style="height:20px;width:211px;">Tuffier retractor</td>
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			<td height="20" style="height:20px;width:211px;">Mosquito forceps</td>
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			<td style="width:119px;">162-7-10</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:211px;">Kelly forceps-curved (14cm)</td>
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			<td style="width:119px;">162-7-38</td>
			<td style="width:167px;"></td>
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		<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>
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			<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>
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		<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>
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			<td height="20" style="height:20px;width:211px;">Needle Drivers</td>
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			<td height="40" style="height:40px;width:211px;">Iris scissors</td>
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			<td style="width:119px;">14058-11</td>
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			<td height="20" style="height:20px;width:211px;">Circulating water pump</td>
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			<td style="width:119px;">J-783X</td>
			<td style="width:167px;"></td>
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		<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>
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		<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>
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		<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>
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			<td height="40" style="height:40px;width:211px;">Penicillin-Streptomycin</td>
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			<td height="40" style="height:40px;width:211px;">EDTA</td>
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			<td style="width:119px;">60-00-4</td>
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		<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>
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			<td height="60" style="height:60px;width:211px;">Azaperone</td>
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			<td style="width:119px;"></td>
			<td style="width:167px;">Stresnil</td>
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			<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>
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			<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>
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			<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>
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			<td height="100" style="height:100px;width:211px;">Bupenorphine hydrochloride</td>
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			<td style="width:167px;">DIN:02347510</td>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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 ...

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

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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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex procedures. The high-quality 3-D visual, multijoint arm and natural tremor filtration allow the surgeon to expose and dissect more accurately with minimal invasion. Total mesorectal excision is the standard surgical technique for the treatment of resectable rectal cancer. To reduce the lateral recurrence rate, lateral pelvic lymph node dissection can be performed, as it is a safe and feasible procedure for locally advanced middle-low rectal cancer with a high possibility of metastasis to the lateral lymph nodes. However, the complexity of the anatomic structures and the high postoperative complication rate limit its application. Recently, several surgeons have increasingly used robotic techniques for total mesorectal excision and lateral pelvic lymph node dissection. Compared with open and laparoscopic surgery, the robotic technique has several advantages, such as less blood loss, fewer blood transfusions, minimal trauma, shorter postoperative hospitalization, and quicker recovery. A robotic approach is generally regarded as a reasonable alternative for complicated procedures such as lateral pelvic lymph node dissection, although there are a limited number of high-quality prospective randomized controlled studies reporting direct evidence. Here, we provide the detailed steps of robot-assisted total mesorectal excision and lateral pelvic lymph node dissection performed at the First Affiliated Hospital of Xi&#39;an Jiaotong University.

The robotic technique described herein aims to detail a stepwise approach to robot-assisted total mesorectal excision and lateral pelvic lymph node dissection for locally advanced (T3/T4) rectal cancer located below the peritoneal reflection.

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex ...