mesenteric lymph duct cannulation in a rat model to harvest lymph fluid

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

On the day before the surgery, cut the cannulas to the appropriate lengths, and use a sterile surgical blade to make a bevel at the tip of each piece of tubing. Next, to prepare the intraduodenal cannula, loop the cannula back onto itself, and then, heat the cannula to create an anchor point in the tubing.
On the day of the surgery, shave the fur from the right side of the abdomen and neck and left clavicle regions of an anesthetized adult rat, and then, clean the surgical regions aseptically with three successive povidone-iodine solution and 70% ethanol scrubs.
To cannulate the mesenteric lymph duct, place the animal with its right side facing up and use a sterile scalpel blade to open the top layer of the abdominal muscle wall with a straight four-centimeter incision extending from the xiphoid process to the right flank, approximately two centimeters below the costal margin.
Retract the small intestine under the left abdominal wall, and use two to three pieces of sterile gauze saturated with normal saline to keep it in place. Then, bridge the rat over a 10-milliliter plastic syringe placed horizontally under the animal&#39;s back at the level of the right kidney to assist in the visualization of the mesenteric lymph duct.
Locate the superior mesenteric lymph duct, which is approximately 0.5 to 1 millimeter in diameter, perpendicular to the right kidney, and immediately rostral and parallel to the dark red pulsating mesenteric artery. Note that in non-fasted rats, as shown here, the superior mesenteric lymph duct is white, opaque, and easier to visualize than in fasted rats.
Next, pass a pair of straight forceps through the peri-renal fat bed at the lower margin of the right kidney parallel to the superior mesenteric lymph duct, and through the connective tissue layers, immediately below the vena cava. Then, using the tip of the forceps, pull the lymph cannula through the peri-renal fat bed with one end immediately adjacent to the mesenteric lymph duct, and the other exteriorized from the animal at the level of the right kidney.
Then, using a surgical microscope and jeweler&#39;s forceps, isolate the mesenteric lymph duct through the overlying layers of connective and fat tissue by blunt dissection, starting at the lower end and taking care not to damage the lymph duct. When the duct has been isolated, use the tip of the fine-tip forceps to make a small hole in the lymph duct. After ensuring that the lymph cannula is completely filled with anticoagulant solution and no air gaps, use a small pair of forceps to insert the lymph cannula approximately two to four millimeters inside the mesenteric lymph duct via the puncture.
If the cannulation is successful, a gradual flow of intestinal lymph will be observed from the free-collecting end of the cannula. When this occurs, place a small drop of cyanoacrylate glue over the entrance hole into the lymph duct to secure the cannula, taking care not to occlude the vessel with excess glue.

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Nanyang Technological University

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JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Assay and Analysis

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Cannulation of the Mesenteric Lymph Duct
<ol>
	<li>Place the animal with its right side facing toward the operator. Perform the surgery with or without the assistance of a surgical microscope as required.</li>
	<li>Open the top layer of the abdominal muscle wall with a straight 4 cm incision extending from the midline (xiphoid process) to the right flank approximately 2 cm below the ribcage (costal margin) using a sterile scalpel blade (see Figure 1B).</li>
	<li>Open the remaining layers of the abdominal muscle wall from 4 - 5 mm lateral to the midline to the right flank with a small pair of surgical scissors (see Figure 1B).</li>
	<li>Retract the small intestine under the left abdominal muscle wall and keep it in place using 2 - 3 pieces of sterile gauze saturated with normal saline.</li>
	<li>Bridge the rat over a 10 ml plastic syringe placed horizontally under the rat&#39;s back at the level of the right kidney to ease visualization of the mesenteric lymph duct.</li>
	<li>Locate the superior mesenteric lymph duct: a vessel approximately 0.5 - 1 mm in diameter that is perpendicular to the right kidney and immediately rostral and parallel to the mesenteric artery (a pulsating dark red blood vessel; see Figure 1C). 
	NOTE: In non-fasted or oil-pre-dosed rats the vessel is white, opaque, and easier to visualize than in fasted rats in which it is quite translucent.</li>
	<li>Inspect the area immediately caudal to the large superior mesenteric lymph duct and the mesenteric artery to determine if a second, smaller accessory lymph duct is present. If present, block the flow of lymph through the duct by severing the duct and occluding with superglue, or tying a suture around the duct, to ensure that the entire volume of lymph flowing from the intestine is collected from the superior mesenteric lymph duct.</li>
	<li>Pass a pair of straight forceps through the peri-renal fat bed at the lower margin of the right kidney, and through the connective tissue layers immediately below the vena cava, in a direction parallel with the superior mesenteric lymph duct.</li>
	<li>Using the tip of the forceps take hold of the lymph cannula and pull it through the peri-renal fat bed with one end immediately adjacent to the mesenteric lymph duct and the other exteriorized from the animal at the level of the right kidney.</li>
	<li>Isolate the mesenteric lymph duct from overlying layers of connective and fat tissue by blunt dissection. Take care not to pierce or damage the lymph duct.</li>
	<li>After isolating the lymph duct, make a small hole in the lymph duct with either micro-scissors, the sharp tip of the jeweler&#39;s forceps, or a 25 G needle. Take care not to completely sever the vessel.</li>
	<li>Ensure that the lymph cannula is completely filled with an anti-coagulant solution (using a syringe and 25 G needle) with no air gaps. Insert the lymph cannula approximately 2 - 4 mm inside the mesenteric lymph duct via the small hole with the aid of a small pair of forceps.</li>
	<li>Observe the lymph cannula for a couple of minutes. Observe a gradual flow of intestinal lymph from the free-collecting end of the cannula if the cannulation is successful. If the cannulation is not successful, repeat steps 1.8 - 1.12.</li>
	<li>If the cannulation is successful, secure the cannula by placing a small drop of veterinary adhesive over the entrance hole into the lymph duct. Take care not to occlude the vessel via the application of excess veterinary adhesive.</li>
	<li>Whilst waiting for the veterinary adhesive to set, observe the flow of lymph for several minutes to ensure that the cannulation procedure was successful. Once certain of the success of the cannulation, remove the gauze pieces that were placed in the abdominal cavity carefully and place the small intestine in its original position.</li>
	<li>Pre-weigh lymph collection tubes, label tubes for lymph and blood sample collection and add anti-coagulant solution. Add sufficient anti-coagulant solution to achieve a final concentration of 1 mg/ml EDTA in sterile water or 10 - 20 IU/ml heparin in the collected lymph or blood.</li>
	<li>Place a lymph collection tube containing an anti-coagulant (step 1.16) at the end of the cannula to collect the free-flowing lymph.</li>
	<li>Next, cannulate the duodenum for infusion of rehydration solutions and/or formulations.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sterile saline</td>
			<td>Baxter healthcare</td>
			<td>AHB 1307</td>
			<td>Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50</td>
		</tr>
		<tr>
			<td>70 % ethanol in water</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Chlorhexidine gluconate solution (Microshield 4)</td>
			<td>Livingstone International</td>
			<td>JJ60243L</td>
			<td>Any brand can be used. http:// www.livingstone.com.au/? PG=search_result&#38;CAT=6&#38;search =JJ60243L</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Livingstone International</td>
			<td>BU0510</td>
			<td>Any brand can be used. http:// www.livingstone.com.au/? PG=search_result&#38;CAT=6&#38;search =BU0510</td>
		</tr>
		<tr>
			<td>Ilium Ketamil (Ketamine 100 mg/ ml)</td>
			<td>PROVET VICTORIA</td>
			<td>KETA I 1</td>
			<td>http://www.provet.vom.au</td>
		</tr>
		<tr>
			<td>Heparin (35000I.U. in 35 mL</td>
			<td>SigmaPharmaceuticals</td>
			<td>337220</td>
			<td>http://www.sigmaco.com.au</td>
		</tr>
		<tr>
			<td>Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm</td>
			<td>Microtube extensions</td>
			<td>PE8050</td>
			<td>Any brand can be used. Example here is PE tubing 0.96 x0.58 mm, 30 m</td>
		</tr>
		<tr>
			<td>Polyethylene (PE) cannula o.d. 0.8 
			mm x i.d. 0.5 mm</td>
			<td>Microtube extensions</td>
			<td>PE9658</td>
			<td>Any brand can be used. Example here is PE tubing 0.8 x0.5 mm, 30 m</td>
		</tr>
		<tr>
			<td>Markers</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Veterinary adhesive</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>23 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN23GX0.75LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http:// www.livingstone.com.au/? PG=search_result&#38;CAT= 6&#38;search=DN23GX0.75LV</td>
		</tr>
		<tr>
			<td>25 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN25GX1.0LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http:// www.livingstone.com.au/? PG=search_result&#38;CAT=6&#38;search= DN25GX1.0LV</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS01TA</td>
			<td>Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http:// www.livingstone.com.au/? PG=search_result&#38;CAT=6&#38;search =T3SS01TA</td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS10SA</td>
			<td>Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http:// www.livingstone.com.au/? PG=search_result&#38;CAT=6&#38;search =T3SS10SA</td>
		</tr>
		<tr>
			<td>Gauze swabs</td>
			<td>Livingstone International</td>
			<td>GSC075</td>
			<td>Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http:// www.livingstone.com.au/? PG=search_result&#38;CAT=6&#38;search =GSC075</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Ratek</td>
			<td>WT1</td>
			<td>Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Zeiss</td>
			<td>495005-0014-000</td>
			<td>Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https:// www.micro-shop.zeiss.com/? l=en&#38;p=us&#38;f=e&#38;i=10143</td>
		</tr>
		<tr>
			<td>1 x Small surgical scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>14060-09</td>
			<td>Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/ Special-Pages/Products.aspx? ProductId=40&#38;CategoryId=17</td>
		</tr>
		<tr>
			<td>2 x Forceps with serrated curved tip</td>
			<td>Fine Science Tools (FST)</td>
			<td>11001-13</td>
			<td>Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/ Special-Pages/Products.aspx? ProductId=405&#38;CategoryId=32</td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/21759/21759_Figure1.jpg" /> 
Figure 1. Photographs of (A) the abdomen shaved in preparation to cannulate the lymph duct, (B) the abdominal muscle wall opened with a straight 4 cm incision extending from the midline to the right flank to access the lymph duct, and (C) the superior mesenteric lymph duct (in yellow circle) perpendicular to the right kidney.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a surgically intervened rat with its abdomen exposed.&#160;
Locate the superior mesenteric lymph duct, lying perpendicular to the right kidney and positioned in front and parallel to the mesenteric artery.
Pass straight forceps through the peri-renal fat bed.
Pull a lymph cannula through the peri-renal fat bed, positioning one end adjacent to the lymph duct and allowing the other end to protrude outside the animal&#39;s body.
Next, create a small incision in the lymph duct.
Fill the cannula with an anticoagulant solution and insert it into the lymph duct.
Inside the duct, the lymph fluid contains several biomolecules including clotting factors that trigger a cascade of events leading to clot formation.
However, the anticoagulant binds to the clotting factors, inhibiting their activity.&#160;&#160;
This enables unclotted lymph fluid to flow freely through the cannula.
Apply a veterinary adhesive over the entrance hole to secure the cannula in place.

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Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

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JoVE Journal

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Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and its associated LNs. The CNS-associated lymphatic system is involved in the drainage of CSF macromolecules and meningeal immune cells toward CNS-draining LNs, thereby regulating waste clearance and immune surveillance within CNS tissues. Presented is a novel approach to obtain three-dimensional (3D) and cellular resolution images of CNS-associated lymphatics while preserving the integrity of their circuits within surrounding tissues. The iDISCO+ protocol is used to immunolabel lymphatic vessels in decalcified and cleared whole mount preparations of the vertebral column that are subsequently imaged with light sheet fluorescence microscopy (LSFM). The technique reveals the 3D structure of the lymphatic network connecting the meningeal and epidural spaces around the spinal cord to extravertebral lymphatic vessels. Provided are 3D images of the drainage circuits of molecular tracers previously injected into either the CSF via the cisterna magna or the thoracolumbar spinal parenchyma. The iDISCO+/LSFM approach brings unprecedented opportunities to explore the structure and function of the CNS-associated lymphatic system in neurovascular biology, neuroimmunology, brain and vertebral cancer, or vertebral bone and joint biology.

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

使用 iDISCO + 和光片荧光显微镜对脊椎淋巴血管和+排水进行三维成像

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and ...

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat&#39;s recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Detailed microsurgical techniques are demonstrated to establish a longer-term jugular vein cannulation rat model for sequential blood collection in the same animal. Physiological and hematological parameters have been monitored during the rat's recovery phase. This model has been applied to study pharmacokinetics of orally administered polyphenol without inducing animal stress.

建立大鼠永久性颈静脉插管的显微外科技能，用于口服药物的连续血液采样

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, ...

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

成績單

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