The authors would like to acknowledge the Cen.Ri.S. (Centro di ricerche sperimentali) of Universit&#224; Cattolica del Sacro Cuore in Rome for permits.

The studies described in this manuscript were approved by the local animal Ethics Committee (Universit&#224; Cattolica del Sacro Cuore, Roma) and were conducted in accordance with the Italian Ministry of Health.
1. Preparation of the cannula for insertion into the proximal branch of the SMA
<ol>
	<li>Cut the larger cannula of 0.93 mm O.D, 0.5 mm I.D. to the required length (about 30 cm).</li>
	<li>Cut the smaller cannula (0.4 mm O.D, 0.25 mm I.D.) to about 5 cm in length and insert it 1 cm into the larger cannula.</li>
	<li>Fix the two cannulas together by cyanoacrylate glue, avoiding occlusion of the lumen.</li>
	<li>Connect the free extremity of larger cannula to a Luer stub adapter (23 G) mounted on a 1 mL syringe filled with saline solution.</li>
	<li>Sharpen the free tip of the smaller cannula with scissors to facilitate insertion of the catheter in the branch of the SMA.</li>
	<li>Check the patency of the cannula by flushing with saline solution. 
	&#8203;NOTE: The sharp end of the cannula will not damage the artery during the animal&#39;s movement because it will be fixed and will not slide along the vessel.</li>
</ol>
2. Preparation of the rat for the surgical procedure
<ol>
	<li>Perform intramuscular anaesthesia with ketamine/xylazine (100/10 mg/kg). 
	NOTE: Sufficient anaesthetic depth is judged by the absence or near-absence of the paw-pinch reflex.</li>
	<li>Shave the fur from the surgical regions: the abdomen for the branch of the SMA cannulation, and the back of the neck for the cannula exit.</li>
	<li>Clean the surgical regions aseptically using a surgical the paw pinch reflex. 
	NOTE: All preparation should be performed with aseptic technique.</li>
	<li>scrub or solution applied in a circular motion, followed by sterile saline or 70% ethanol, 3 times.</li>
	<li>Place the animal in a supine position, immobilizing the four limbs.</li>
</ol>
3. Cannulation of a proximal branch of the SMA
<ol>
	<li>Ensure proper anesthetic depth by testing the paw pinch reflex prior to incision.</li>
	<li>Apply a sterile surgical, water-resistant drape.</li>
	<li>With a scalpel blade, open the abdominal wall with a straight 3 cm incision on the midline of the mesogastric region through all the abdominal planes into the peritoneum.</li>
	<li>Place gauzes, soaked with saline solution, around the laparotomy incision on top of the surgical drape. Use sutures to keep the surgical incision open. 
	NOTE: All swabs and surgical instruments must be sterile.</li>
	<li>Use cotton swabs to identify and expose the small intestine. Follow its natural disposition to identify the mesentery. Extract the mesentery out of the laparotomic cut and lay it downward on the gauzes (Figure 1A).</li>
	<li>Identify the SMA by feeling the pulsation.</li>
	<li>Use the cotton swabs to &#34;make way&#34; between the mesenteric fat and uncover the SMA and 2-3 of its proximal branches.</li>
	<li>Choose a proximal branch of the SMA sufficiently large to allow the surgical maneuvers of cannulation. Tie this branch (with a 4-0 silk suture) 3-4 cm downstream from its origin to allow its expansion keeping the suture ends long enough to be manipulated later.</li>
	<li>Place a rigid support under the branch of the SMA. The handle of the surgical forceps is sufficient here.</li>
	<li>Hold the extremity of the smaller cannula (linked with the bigger cannula at the opposite extremity) with the dominant hand using forceps and pull the suture ends with the other hand to strain the vessel and to facilitate the entry of the catheter (Figure 1B).</li>
	<li>Hold the tip of the cannula at a 20&#176; angle from the plane of the vessel in the direction opposite to the blood flow.</li>
	<li>Lightly press the tip to penetrate the artery wall and insert the cannula. 
	NOTE: Cannulation is performed without cutting the artery; the tip of the catheter will break the vessel wall and facilitate the entry. Blood flowing back into the cannula confirms the correct insertion.</li>
	<li>Continue the insertion of the cannula for another 1 cm in the arterial branch close to the origin from the SMA.</li>
	<li>Fix the cannula to the artery with a surgical knot (4-0 silk) and verify its correct functioning by flushing 1 mL of sterile saline solution or with a blood sampling.</li>
</ol>
4. Tunneling of the cannula and placement in the infusion support system
<ol>
	<li>Place a sterile surgical drape on the incision prior to changing the animal&#39;s position. 
	NOTE: Tunnel from back to abdomen is created by exerting pressure in the subcutaneous space with a pointed surgical instrument. A sterile surgical drape on the abdomen and back incisions should be used.</li>
	<li>Make a 1 cm incision of the posterior region of the neck and accommodate a spherical valve.</li>
	<li>Pass the cannula from the laparotomy access to the valve placed in the neck through subcutaneous tissues (Figure 2A). Close the cannula distal extremity with a catheter plug to avoid air inflow.</li>
	<li>Replace the small bowel in the abdominal cavity. Close the abdominal wall and close the skin incisions with a continuous 3-0 silk suture.</li>
	<li>Secure the valve to the neck skin with stitches.&#160;Close the skin incisions with a continuous 3-0 silk suture.</li>
</ol>
5. Post-operative management
<ol>
	<li>Dress the rat with a jacket to protect the button valve. Protect the exposed part of the cannula with a steel rod during infusion and secure it to the valve (Figure 2B). 
	NOTE: Since surgery is performed under aseptic technique, antibiotics are not indicated. NSAID should be administered pre-operatively for pain control (5 mg/mL Meloxicam injectable, 1 mg/kg once daily for up to 3 days). After operation, stabilize the rat in a metabolic cage for the time of infusion (24h). Then re-anesthetize the rat with isoflurane inhalation for the time necessary to disassemble the infusion system. Subsequently it is possible to house the rat in a normal cage with a 12-hour light/dark cycle and free access to food and water.</li>
	<li>Stabilize the rat in a metabolic cage for the time of infusion. The rat is now awake and free to move and eat in the cage.</li>
	<li>Connect the distal extremity of the cannula to an elastomeric pump (100 mL volume max, 5.0 mL/h flow rate) filled with 50 mL of sterile saline solution. Proceed with infusion for 24 hours (Figure 2C).</li>
	<li>On the first day, administer intramuscular antibiotics (enrofloxacin 10 mg/kg for the first 24 h) and then pass to oral administration (enrofloxacin 100 mg in 500 mL in drinking water). Dispense analgesic therapy intramuscularly during awakening (ketoprofen 5.0 mg/kg) and in the following days orally (paracetamol 200 mg in drinking water). 
	NOTE: Dilute oral therapy administered in drinking water to obtain a bearable taste. Monitor body weight measurement and hydration.</li>
	<li>At the end of the infusion time (24 hours) disassemble the animal&#39;s external infusion system by removing the pump, the jacket, the steel rod, and the valve from the rat. Close and cut the cannula as it comes out of the neck, leaving this extremity under the skin of the neck after wound suture. 
	NOTE: In this phase it may be necessary to anesthetize rats for few minutes by isofluorane inhalation.</li>
	<li>House the rat, individually, in a normal cage with a 12-hour light/dark cycle and free access to food and water. 
	NOTE: Baseline post-op food intake is about 30 g./day Baseline water intake is about 50 ml/day. Average&#160;weight should be about 400 mg.</li>
</ol>

<ol>
	<li>Zhang, Z., Chen, X., Zhu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+mechanical+thrombectomy+treatment+of+acute+superior+mesenteric+artery+embolism.">Percutaneous mechanical thrombectomy treatment of acute superior mesenteric artery embolism.</a> European Journal of Vascular and Endovascular Surgery Short Reports. 34, 17-20 (2017).</li><li>Wang, M. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transradial+approach+for+transcatheter+selective+superior+mesenteric+artery+urokinase+infusion+therapy+in+patients+with+acute+extensive+portal+and+superior+mesenteric+vein+thrombosis.">Transradial approach for transcatheter selective superior mesenteric artery urokinase infusion therapy in patients with acute extensive portal and superior mesenteric vein thrombosis.</a> Cardiovascular and Interventional Radiology. 33 (1), 80-89 (2010).</li><li>Zammit, M., Toledo-Pereyra, L. H., Malcom, S., Konde, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cranial+mesenteric+vein+cannulation+in+the+rat.">Long-term cranial mesenteric vein cannulation in the rat.</a> Laboratory Animal Science. 29 (3), 364-366 (1979).</li><li>Aguiar, J. L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+long-term+infusion+into+the+inferior+mesenteric+artery+of+unrestrained+rats.">Technique for long-term infusion into the inferior mesenteric artery of unrestrained rats.</a> Laboratory Animals. 22 (2), 173-176 (1988).</li><li>Trevaskis, N. L., Hu, L., Caliph, S. M., Han, S., Porter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mesenteric+lymph+duct+cannulated+rat+model:+application+to+the+assessment+of+intestinal+lymphatic+drug+transport.">The mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport.</a> Journal of Visualized Experiments. (97), e52389 (2015).</li><li>Leivestad, O., Malt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+infusion+into+the+hepatic+artery+and+vena+cava+of+the+rat.">Continuous infusion into the hepatic artery and vena cava of the rat.</a> Surgery. 74 (3), 401-404 (1973).</li><li>Eloy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+vascular+perfusion+of+the+isolated+rat+small+bowel.+Importance+of+the+intestinal+brush+border+enzyme-release+in+basal+conditions.">Ex vivo vascular perfusion of the isolated rat small bowel. Importance of the intestinal brush border enzyme-release in basal conditions.</a> European Surgical Research. 9 (2), 96-112 (1977).</li><li>Liu, R. N., Wei, X. J., Li, S. P., Jiang, C., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+invasive+dynamic+blood+pressure+between+superior+mesenteric+artery+and+common+carotid+artery+in+rats.">Comparison of invasive dynamic blood pressure between superior mesenteric artery and common carotid artery in rats.</a> World Journal of Emergency Medicine. 11 (2), 102-108 (2020).</li><li>Leung, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+mesenteric+artery+is+more+important+than+inferior+mesenteric+artery+in+maintaining+colonic+mucosal+perfusion+and+integrity+in+rats.">Superior mesenteric artery is more important than inferior mesenteric artery in maintaining colonic mucosal perfusion and integrity in rats.</a> Digestive Diseases and Sciences. 37 (9), 1329-1335 (1992).</li><li>Gui, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+artery+botulinum+toxin+(BoNT/A1)+infusion+selectively+blocks+bowel+peristalsis+in+rats.">Mesenteric artery botulinum toxin (BoNT/A1) infusion selectively blocks bowel peristalsis in rats.</a> Journal of the American Chemical Society. 231 (4), 19-20 (2020).</li><li>Lecomte, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+gut+microbiota+in+rats+fed+a+high+fat+diet+correlate+with+obesity-associated+metabolic+parameters.">Changes in gut microbiota in rats fed a high fat diet correlate with obesity-associated metabolic parameters.</a> PLoS One. 10 (5), 0126931 (2015).</li><li>Hajj Hussein, I. 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The authors have nothing to disclose.

The main advantage of this rat SMA infusion model is its steadfastness and durability for at least 24 hours in the vast majority of the animals. The infusion of anti-coagulant might lengthen this time interval. The model allows a reliable infusion of drugs selectively in the mesenteric region, targeting the small bowel and the proximal part of the colon.
Several steps are critical to the success of the technique. To achieve cannulation in a very small vessel it is important to select rats weighing at least 400 g; the sex and age are not relevant. It is also important to choose the correct surgical instruments and the type of cannula. Here, a smaller polyurethane cannula (0.4 mm O.D, 0.25 mm I.D.) is inserted 1 cm into the larger cannula (0.93 mm O.D, 0.5 mm I.D.) to obtain a functional and useful catheter to allow both connections to the small artery and to the larger infusion system.
The first surgical critical step is cleaning the SMA and the branch identified for cannulation from the surrounding adipose tissue (step 3.5). This helps avoid the insertion of the cannula between the tissue and the artery, which is a common mistake. However, this cleaning step is difficult as the little branch of the SMA is fragile and easy to damage. If the branch is injured, it is possible to stop the bleeding by ligature and to choose a different proximal branch, as to not to waste the animal.
To prevent air bubble formation within the cannula and avoid gas embolism, the cannula must be filled with saline until the tip before insertion in the branch. To secure the cannula in place, the application of surgical thread (4-0 silk) must be between the point of insertion into the artery and the cannula tip, directly on top of the vessel around the catheter. The surgical knot must be tight enough to fix the cannula but not too tight to occlude it (step 3.12).
The best way to ensure a correct cannulation is to see blood flow back through the cannula (step 3.10). In terms of troubleshooting, if this does not take place, it may be due to the following reasons:
the cannula was not correctly inserted into the artery;
the cannula is inside the artery but occluded by the node in an incorrect position;
the cannula is inside the artery and an air bubble in the cannula is slowing down the flow;
a clot has formed within the cannula.
An incorrect insertion may be due to cannula positioning in the space between the artery and the adipose tissue. In this case re-insertion is necessary. When the knot&#160;above the vessel occludes the cannula, it is possible to untie it very carefully and remake it. Small air bubbles in the catheter generally do not compromise the cannulation and are not life-threatening; but if there is a big air bubble in the cannula it is necessary to draw back on the cannula using the syringe or re-position&#160;the catheter in a different branch. Usually, it is possible to avoid clot formation and keep the cannula patent by infusing 0.2 mL boluses of saline once in a while during operation.
A limitation of this study is an under-evaluation of the patency of the cannula in longer infusion times: here, a 24 hour infusion was performed while rats where housed in a metabolic cage. To obtain a longer infusion period, it may be useful to use anti-coagulant therapy, not administered in this study. However, during infusion, the rat must be housed in the metabolic cage because it is the only one that supports the infusion system. This location is uncomfortable for the animal which might be stressed if treated for a longer period. Furthermore, only saline solution was used for infusion, so there are no results about specific drug administration. One limitation of the method is the impossibility to infuse in the arterial branches (if present) above that used for the catheter. For this reason it is recommended to cannulate the closest branch from the aorta.
No other rat SMA long-term infusion model for unrestrained animals is present in literature. Compared to the IMA cannulation model described many years ago4, the described technique here has a wider experimental target because it allows drug infusion in the SMA perfusion area and is not limited to the colon. Recently, for the first time, selective cannulation of a branch of the SMA was used for infusion of botulinum toxin directly in the arterial mesenteric region to study the effect on the intestinal smooth muscle10, but many other drugs could be tested in future. For example, anticoagulants can be infused to study mesenteric thrombosis, or drugs with an intestinal microbiota action11 or even drugs for inflammatory bowel diseases12. Intra-arterial infusion is useful for intestinal metabolism studies in particular, because the drug effect is evaluable before the blood goes through the portal circulation where it is subject to hepatic metabolism.

The superior mesenteric artery (SMA) in humans as in rats originates from the abdominal aorta and supplies the bowel with arterial blood from the duodenum to the proximal transverse colon. SMA gives rise to numerous branches.
After capillary perfusion, the mesenteric circulation is drained through the portal vein to the liver, where it undergoes hepatic metabolism before being readmitted to the systemic circulation. Cannulation of the SMA is useful for diagnostic purposes, therapeutic embolization and drug infusion in a selective or continuous manner to evaluate the effect on the bowel or, most importantly, the liver metabolism and chemical clearance. In humans, minimally invasive radiological catheterization of the SMA is performed for endovascular treatment1 or selective drug infusion2 using several percutaneous approaches like transfemoral or transaxillary puncture and cannulation.
There are literature reports of different techniques of cannulation of the small abdominal vessels: the superior mesenteric vein (SMV)3, the inferior mesenteric artery (IMA)4, the mesenteric lymph duct5, the hepatic artery6 or studies for ex vivo on bowel perfusion7 in rats. In comparison with the venous side, cannulation of the SMA in rats is much more demanding because of the simultaneous risks of thrombosis and bleeding, provided its high pressure. In particular, problems arise in case the cannulation is in operation when the rat awakes from anaesthesia on the surgical bed and more if the experiment requires a free animal in a cage after surgery.
A recent paper has described SMA cannulation as a part of the experiment (blood pressure measure) in an animal under anaesthesia8. However, no technique is described on the surgical cannulation of the SMA for long-term infusion in an unrestricted animal. The aim of this manuscript is to describe step by step a surgical technique for long term cannulation of the SMA through a proximal branch, which allows for the selective infusion of drugs into the mesenteric bed for at least 24 hours (and over). As a steady and sturdy cannulation requires permanent ligation and closure of the vessel where the catheter is inserted, this technique instead avoids inserting the catheter directly into the SMA9 and approaches the vessel through the cannulation of a proximal branch, as proximal as possible to the very origin of the SMA from aorta. Proximal infusion allows the infused drug to reach the widest anatomic bed possible, without closing the blood flow through the principal vessel.
The rat SMA cannulation technique has many applications. It would be possible to administer drugs selectively in the mesenteric arterial compartment to obtain local action at the gastrointestinal level and to avoid systemic effects and hepatic drug metabolism. The SMA-cannulated rat model has advantages over larger animal models: it is less expensive, it is ethically acceptable, and it is easier to perform and learn. SMA cannulation surgery is also easier to perform in the rat model compared to the mouse model.

<table><tbody><tr><td>Crile-Wood Needle Holder</td><td>2Biological Instruments</td><td></td><td>Tip Shape: Straight; Tip Width: 2 mm; Clamping Length: 14 mm; Lock: Yes; Scissors: No; Alloy / Material: Stainless Steel; Length: 15 cm; Serrated: Yes</td></tr><tr><td>Extra Fine Graefe Forceps</td><td>2Biological Instruments</td><td></td><td>Tip Width: 0.5 mm; Tip Dimensions: 0.5 x 0.5 mm; Alloy / Material: Stainless Steel; Length: 10 cm</td></tr><tr><td>Luer Stub Adapter</td><td>BD Intramedic</td><td></td><td>23 gauge for use with 427410 tubing</td></tr><tr><td>Membrane valve</td><td>Biomed</td><td>Mod 617</td><td></td></tr><tr><td>Poliurethane Catheter</td><td>ENKI</td><td></td><td>external diameter: 0.4 mm, internal diameter: 0.25 mm</td></tr><tr><td>Silastic Catheter Laboratory tubing</td><td>Healthcare industries</td><td>508-002</td><td></td></tr><tr><td>Spring Scissors</td><td>2Biological Instruments</td><td></td><td>Tip Shape: Angled; Tips: Sharp; Alloy / Material: Stainless Steel</td></tr><tr><td>Student Surgical Scissors</td><td>2Biological Instruments</td><td></td><td>Tip Shape: Straight; Alloy / Material: Student Stainless Steel; Serrated: No; Feature: Student Quality</td></tr></tbody></table>

targeting the rat's small bowel: long-term infusion into the superior mesenteric artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

In this study, the procedure was performed on 15 rats. At the end of 24 hours of saline infusion, no signs of saline or blood loss have been observed in the metabolic cages and the abdominal wound was clean in all animals as were the cages.
In the normal cages, rats were observed for 5 days with daily monitoring of weight and water/food intake. During this period, the general condition of the animals at gross examination was good with no indications of behavioral abnormalities. All rats immediately after surgery started feeding again. The average daily food and water intake increased progressively until normal after 3 days as shown in Figure 3A and 3B, respectively. In Figure 3C, it is possible to see that weight gain was regular, gradually increasing until the end of the observation period. No alterations of bowel movements took place and daily feces and urine output were normal.
After 24 hours, there was saline residue (respectively, 40 mL and 20 mL) in only 2 pumps filled with 50 mL of saline solution while all others (86.7%) were empty. Furthermore, after this infusion period, 12 cannulas (80%) were still functional for both blood sampling and saline infusion (5 mL), while 3 cannulas were not patent anymore (2 of these were the cannulas connected to the pumps with residue) (Table 1).
At necropsy, 100% of cannulas (n=15) were still located in the SMA branch and no rats had signs of bowel ischemia (Figure 4B) or intrabdominal bleeding. The 3 occluded cannulas were found kinked respectively at 0.5 cm, 1 cm and 1.5 cm from the insertion in the SMA branch. This phenomenon is probably due to the movements of the animals in the cages.
In 5 rats, immediately after the procedure and before pump connection, 2 mL of iodinated contrast medium were injected into the mesenteric cannula, to obtain an angiography through an image intensifier (angiography was performed intraoperatively). In each rat (n=5), it was possibile to see the mesenteric arterial circle and the SMA and its main branches without contrast medium spreading in the abdomen as shown in Figure 4A. This confirmed that the cannula was well placed and fixed to the branch of the SMA.
<img alt="Surgical technique and intestinal repair in medical procedure, showing suturing close-up." class="xfigimg" src="/files/ftp_upload/60787/60787fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Experimental photographs. (A) The small intestine following its natural disposition on a gauze (it is possible to visualize the SMA with all the branches); (B) The operator inserting the cannula into the SMA branch. It is necessary to have a solid support under the vessel to guarantee the insertion of the tube. The distal silk suture closes the vessel and the proximal one fixes the catheter inside the branch. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60787/60787fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rat infusion experiment setup; surgical cannula placement, infusion, and container diagram." class="xfigimg" src="/files/ftp_upload/60787/60787fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Infusion support system. (A) Once tunneled subcutaneously, the cannula emerges from the posterior region of the neck through the white valve; (B) A rat wearing a jacket to stabilize the white valve. A steel rod protects the catheter during infusion. (C) Diagramatic representation of a rat housed in a metabolic cage during infusion of saline with an elastomeric pump connected to the cannula exiting the steel rod. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60787/60787fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Food, water intake, and weight gain graphs over 5 days, showing trend analysis, data comparison." class="xfigimg" src="/files/ftp_upload/60787/60787fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Representative data for food intake, water intake and weight gain of rats (n=15) in observation period of 5 days. The average daily food (A) and water (B) intake progressively increases, and it stabilizes at physiological levels after 3 days. The average weight gain (C) gradually increases until the end of the observation period. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60787/60787fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluoroscopy image of catheter placement; surgical view of abdominal exploration for catheter insertion." class="xfigimg" src="/files/ftp_upload/60787/60787fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Photographs of (A) the contrast angiography of the mesenteric arterial region after contrast infusion through the cannula (proof of adequately placed cannula) and (B) the cannula still well positioned during autopsy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60787/60787fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Elastomeric pump</td>
			<td colspan="2">Cannula</td>
		</tr>
		<tr>
			<td></td>
			<td>Empty</td>
			<td>With residue</td>
			<td>Patent</td>
			<td>Not Patent</td>
		</tr>
		<tr>
			<td>n=15</td>
			<td>13</td>
			<td>2</td>
			<td>12</td>
			<td>3</td>
		</tr>
		<tr>
			<td>%</td>
			<td>86.7</td>
			<td>13.3</td>
			<td>80</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 1: Elastomeric pump dischargeand cannula patency after 24 hours of saline infusion. Patency was tested by drawing blood with a syringe and infusing 5 mL of saline in the cannula.

Watch this Scientific Journal Video about Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery at JoVE.com

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

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JoVE Journal

Bioengineering

4.1K Views.  Università Cattolica del S. Cuore. Superieure mesenterische arterie cannulatie bij de rat is moeilijk en het wordt niet vaak gebruikt als een experimenteel model. Deze techniek maakt het mogelijk om een functionele cannulatie uit te voeren en opent een experimenteerveld met eerste passage-effecten van intra-arteriële geneesmiddelen. Deze methode voorkomt bloedingen en trombose terwijl de cannulatie van de superieure mesenteriale slagader wordt goedgekeurd.En het mogelijk maken van langdurige infusie van medicijnen in e...

Video: Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Medicine

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

Immunology and Infection

Standardized Colon Ascendens Stent Peritonitis in Rats - a Simple, Feasible Animal Model to Induce Septic Acute Kidney Injury

AKI in septic patients is associated with increased mortality and poor outcome despite major efforts to refine the understanding of its pathophysiology. Here, an in vivo model is presented that combines a standardized septic focus to induce AKI and an intensive care (ICU) setup to provide an advanced hemodynamic monitoring and therapy comparable in human sepsis. Sepsis is induced by standardized colon ascendens stent peritonitis (sCASP). AKI is investigated functionally by measurement of blood and urine samples as well as histologically by evaluation of histopathological scores. Furthermore, the advanced hemodynamic monitoring and the possibility of repetitive blood gas sampling enable a differentiated analysis of severity of induced sepsis.
The sCASP method is a standardized, reliable and reproducible method to induce septic AKI. The intensive care setup, continuous hemodynamic and gas exchange monitoring, low mortality rate as well as the opportunity of detailed analyses of kidney function and impairments are advantages of this setup. Therefore, the described method may serve as a new standard for experimental investigations of septic AKI.

Acute kidney injury (AKI) is a severe complication in critically ill patients and is related with an increased mortality. Here, we present a reliable and reproducible in vivo model to mimic AKI under inflammatory conditions that might contribute towards understanding the pathogenesis of septic AKI.

AKI in septic patients is associated with increased mortality and poor outcome despite major efforts to refine the understanding of its pathophysiology. ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biology

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...

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.

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

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

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