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. 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=Inflammatory+bowel+disease+in+rats:+bacterial+and+chemical+interaction.">Inflammatory bowel disease in rats: bacterial and chemical interaction.</a> World Journal of Gastroenterology. 14 (25), 4028-4039 (2008).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;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)&#160;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="Figure 1" class="xfigimg" src="/files/ftp_upload/60787/60787fig01.jpg" /> 
Figure 1:&#160;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/files/ftp_upload/60787/60787fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60787/60787fig02.jpg" /> 
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/files/ftp_upload/60787/60787fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60787/60787fig03.jpg" /> 
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/files/ftp_upload/60787/60787fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60787/60787fig04.jpg" /> 
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/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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Research

JoVE Journal

Bioengineering

4.1K Views.  Università Cattolica del S. Cuore. 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. 

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

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...