Name: basic surgical techniques in the g&ouml;ttingen minipig: intubation, bladder catheterization, femoral vessel catheterization, and transcardial perfusion
Uploaded: 2011-06-26T14:00:00
Duration: 13 min 26 s
Description: Watch this Scientific Journal Video about Basic Surgical Techniques in the GÃ¶ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion at JoVE

The study was financially supported by the Lundbeck Foundation, The Karen Elise Jensen Foundation, The Health Research Fund of Central Denmark Region and The Institute of Clinical Medicine Aarhus University Hospital. We acknowledge with gratitude the skilful assistance of Donald F Smith, Anders Steen Soerensen, Henrik Soerensen, Asbjoern Ettrup-Christensen, and the staff at Paaskehoejgaard.

1. Intubation
<ol>
	<li>Equipment needed for endotracheal intubation: means of assisted ventilation, slings for opening the mouth, low pressure suction with blunt tip, veterinary laryngoscope with straight blade between 17 and 25 cm long, stylet, syringe with air, stethoscope and adhesive tape. For adult G&ouml;ttingen minipigs an endotracheal tube size 5 to 7 mm is appropriate10. In this video a 5.5 mm tube was used 10.</li>
	<li>The minipig is placed on its back and properly supported in order to ensure that the laryngeal passage is kept straight.</li>
	<li>The tongue is pulled out slightly and the jaw held open by an assistant without overextending the head. Rotation of the airways or excessive extension of the head can occlude airways, making the laryngeal opening more difficult to identify and complicating tracheal placement of the tube.</li>
	<li>The laryngoscope is passed into the pharyngeal cavity and used to depress the tongue, making the epiglottis visible. The tip of the suction is used to displace the epiglottis from the soft palate. The tip of the laryngoscope is then used to press the epiglottis upward toward the base of the tongue, revealing the laryngeal opening.</li>
	<li>The arytenoids cartilages and vocal cords come into view and the endotracheal tube is advanced gently with a slight rotation, into the trachea during expiration.</li>
	<li>The tube cuff is inflated according to the specifications of the manufacture while taking care to avoid excess inflation, which may cause swelling and edema of the airways. Pigs have a bronchus to ventilate the right cranial pulmonal lobe. It is therefore important to place the tube above the tracheal bifurcation. This is facilitated by using the correct tube size and importantly by fastening the proximal end of the tube at the level of the snout using adhesive tape.</li>
	<li>When the minipig is properly intubated, a free passage of air must be felt through the tube, and chest auscultation should reveal respiration sounds in both the left and right side of the thorax.</li>
	<li>Upon connecting the ventilator with monitoring system to the minipig, physiological expiratory carbon dioxide values should be measurable and the carbon dioxide curve that follows the respiration pattern decided by the ventilator settings6,10 can be observed.</li>
	<li>The carbon dioxide and oximetry values may initially be affected by the premedication and anesthesia induction. However these values should quickly revert back to normal with proper ventilation6,10. Any sign of cyanosis despite artificial-assisted ventilation indicate improper placement of the tube. The most common error is inadvertent placement of the tube in the esophagus.</li>
</ol>
2. Transurethral bladder catheterization
<ol>
	<li>This procedure is only for female pigs. For adult minipigs a Foley catheter French size 8 or 10 is appropriate. Due to the curvature and size of the external urethral opening, a catheter with stylet is preferable. A nasal speculum can be used in small minipigs whereas a speculum with longer blades can be useful for large animals. Necessary equipment also includes a blunt-tip forceps, a syringe with saline, lubricant and a closed system urine drainage bag.</li>
	<li>In order to best visualize the external urethral orifice during catheterization the minipig is placed on its back with a support under the lumbar region, while an assistant pulls the hind legs cranially.</li>
	<li>The lubricated speculum is inserted and opened to visualize the vaginal wall. The female external urethral opening is located in the floor of the vagina approximately one-third to one-half of the distance to the cervix (Fig. 1A). However, because the animal is placed on the back, the position of the urethral opening will appear to be inverted.</li>
	<li>The catheter is controlled with the blunt-tip forceps, so the tip of the catheter is in continuation of the forceps. Carefully insert the catheter in the urethral opening (Fig. 1B). The urethra is easily traumatized if too much force is used during catheterization. However, slight resistance may initially be felt until the catheter has passed the external urinary sphincter. If this problem persists, this may indicate that the animal is not sedated deeply enough. The solution is to wait awhile and let the anesthesia take its effect. </li>
	<li>As the catheter reaches the bladder, remove the stylet, and urine will be seen flowing. </li>
	<li>Inflate the balloon with saline according to the specifications of the manufacture and gently pull the catheter out until the balloon catches on the bladder neck.</li>
	<li> Attach the closed-system urine drainage bag. </li>
	<li>Tape the catheter to the tail to avoid accidental displacement of the catheter when handling the animal.</li>
</ol>
3. Femoral artery and vein catheterization 
<ol>
	<li>Necessary equipment includes: scalpel, blunt-tip surgical scissors, tissue forceps, small blunt-tip surgical forceps, small self-retaining tissue retractor, needle holder, surgical swaps, suture with needle, heparinized saline as a flush-fluid to maintain catheter patency, two 18-gauge IV cannula, two 4-French brite tip sheaths with introducer and Seldinger guide wire.</li>
	<li>The depth of anesthesia should be assessed by testing the interdigital pain reflex before initiating the procedure. </li>
	<li>The femoral artery and vein are approached with the minipig placed on its back and the rear leg retracted laterally. Identify in the skin fold between the gracilis and sartorius muscle where the pulsation of the superficial part of the medial saphenous artery disappears. </li>
	<li>Make a longitudinal superficial skin incision cranial to this point, thereby avoiding inadvertently damaging the medial saphenous vessels. </li>
	<li>Use a blunt-tip scissors to dissect the underlying subcutaneous tissue. </li>
	<li>The fascia division of the sartorius and gracilis muscle is divided cranial to the penetration site of the medial saphenous vessels, first with a small blunt-tip surgical forceps and then digitally. </li>
	<li>The two muscle groups are separated with a small self-retaining tissue retractor while taking care not to damage the femoral nerve and vessels. </li>
	<li>The artery is isolated for a length of approximately 1 to 2 cm using blunt dissection.</li>
	<li>Rotate the beveled venflon needle so the lumen is facing upwards. Bend the needle a little so it will follow the vessel curvature more easily. Remember to ensure the needle still can move back and forth with little effort.</li>
	<li>The artery is punctured, the needle retracted and the Seldinger guide wire is inserted through the venflon tube.</li>
	<li>Remove the venflon tube while gently applying pressure on the artery to ensure fixation of the guidewire.</li>
	<li>The sheath with introducer is inserted over the Seldinger wire and advanced to the desired position, after which the introducer and Seldinger wire is removed.</li>
	<li>The femoral vein is located just below and medial to the artery. After isolating the vein using blunt dissection, the vein is cannulated as previously described for the artery. </li>
	<li>To verify that both the artery and vein are correctly catheterized, draw blood from both sheaths and compare the color of the samples. Whenever drawing a blood sample, remember to subsequently flush the catheters with heparinized saline in order to maintain catheter patency. </li>
	<li>The catheters are secured and the skin is closed with a few sutures.</li>
	<li>A vessel loop can be placed around the artery and vein in order to fixate the vessels prior to puncture. Despite its advantages, we generally avoid this additional step, because it requires excess dissection of the vessels and can increase the risk of damaging the lateral and deep branches.</li>
</ol>
4. Transcardial perfusion
<ol>
	<li>The following equipment is needed for the procedure: perfusion system (Fig. 2), relevant perfusion fluids, scalpel, blunt-tip surgical scissors, bone shears, and two large self-holding forceps of which at least one has a curved tip. A self retaining sternal retractor is not absolutely necessary, but it eases the procedure considerably. </li>
	<li>Transcardial perfusion must be performed in a well ventilated room with a high power fume extraction system and facilities that allow collection of perfusate liquids and blood for subsequent safe disposal.</li>
	<li>After intravenously injecting a lethal dose of pentobarbital, test the interdigital pain reflex before continuing. </li>
	<li>The best access to the heart and aorta is provided via a median sternotomy. Make a deep longitudinal skin incision, extending from the manubrium to the xiphoid process of the sternum. Gain access to the thoracic cavity by making a small incision in the diaphragm just below the xiphoid process.</li>
	<li>Insert the bone shears into the thoracic cavity and bisect the sternum. Care must be taken when performing this procedure, because the heart may be adherent at some place to the inside of the sternum. Therefore, make certain that the blade of the bone shears rests at all time inside the thoracic cavity against the inside of the sternum. </li>
	<li>After the sternum has been divided, put in the self-retaining sternal retractor and open the thoracic cavity. </li>
	<li>If still intact, make a small incision in the pericardium at the apex of the heart and then digitally open the pericardial sac. </li>
	<li>Identify the left ventricle, the right auricle and the aorta together with the superior caval vein (Fig. 1C)</li>
	<li>Make a small superficial cut near the apex of the heart and access the left ventricle by perforating the myocardium with a blunt-tipped instrument. </li>
	<li>Insert the perfusion cannula inside the left ventricle and move the cannula tip cranially into the aorta. </li>
	<li>When the cannula is felt between the fingers in the aorta, clamp the cannula into place with the self-holding curved forceps. </li>
	<li>Make an incision in the right auricle and begin injecting the fixative through the perfusion cannula. The flow of perfusate in the aorta can be felt, and blood will pour out through the incised right auricle as the perfusate flow into the minipig vessels continues. Notice how systemic perfusion with paraformaldehyde induces hyperextension of the limbs and twitching of the superficial musculature due to aldehyde cross-linkage of nerves and muscles. When using a perfusion system powered by compressed air instead of gravity, make sure to remove the cannula, or clamp the clear silicone tube, before the canister is empty, in order to avoid air from entering the vascular system.</li>
</ol>
<img src="/files/ftp_upload/2652/2652fig1.jpg" alt="Figure 1" /> Figure 1. A: The female G&ouml;ttingen minipig external urethral opening. B: The urethral opening after insertion of catheter. C: Ventral view of the minipig heart after median sternotomy.
<img src="/files/ftp_upload/2652/2652fig2.jpg" alt="Figure 2" /> Figure 2. A: The perfusion system consists of inlet tubing with an adjustable pressure valve and a lever used to control inlet of compressed air into the container holding the perfusate. Clear silicone tubing connects the container to a blunt-tip cannula made from a 4 mm steel pipe. B and C: By compressing the inlet lever, the pressure rises in the container and the perfusate is propelled through the cannula. D: When the clear silicone tubing and cannula are filled with perfusate, the flow is stopped with a clamp and the system is ready for use.

<ol>
	<li>Lind, N. M., Moustgaard, A., Jelsing, J., Vajta, G., Cumming, P., Hansen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pigs+in+neuroscience:+modeling+brain+disorders.">The use of pigs in neuroscience: modeling brain disorders.</a> Neurosci Biobehav Rev. 31, 728-751 (2007).</li><li>Ettrup, K. S., Sorensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+Gottingen+minipig+hypothalamus.">The anatomy of the Gottingen minipig hypothalamus.</a> J Chem Neuroanat. 39, 151-165 (2010).</li><li>Larsen, M. O., Rolin, B., Sturis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+insulin+responses+as+predictive+markers+of+pancreatic+beta-cell+mass+in+normal+and+beta-cell-reduced+lean+and+obese+Gottingen+minipigs+in+vivo.">Measurements of insulin responses as predictive markers of pancreatic beta-cell mass in normal and beta-cell-reduced lean and obese Gottingen minipigs in vivo.</a> Am J Physiol Endocrinol Metab. 290, 670-677 (2006).</li><li>Forster, R., Ancian, P., Fredholm, M., Simianer, H., Whitelaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minipig+as+a+platform+for+new+technologies+in+toxicology.">The minipig as a platform for new technologies in toxicology.</a> J Pharmacol Toxicol Methods. , (2010).</li><li>Popov, A. F., Dorge, H., Hinz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+intimal+hyperplasia+in+aortocoronary+internal+mammary+vein+grafts+in+minipigs.">Accelerated intimal hyperplasia in aortocoronary internal mammary vein grafts in minipigs.</a> J Cardiothorac Surg. 3, 20-20 (2008).</li><li>Swindle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques. , (2007).</li><li>Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., Jorgensen, R. L., Sorensen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+stereotaxic+targeting+in+pigs+based+on+a+stereotaxic+localizer+box+fitted+with+an+isocentric+frame+and+use+of+SurgiPlan+computer-planning+software.">MRI-guided stereotaxic targeting in pigs based on a stereotaxic localizer box fitted with an isocentric frame and use of SurgiPlan computer-planning software.</a> J Neurosci Methods. 183, 119-126 (2009).</li><li>Val-Laillet, D., Blat, S., Louveau, I., Malbert, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computed+tomography+scan+application+to+evaluate+adiposity+in+a+minipig+model+of+human+obesity.">A computed tomography scan application to evaluate adiposity in a minipig model of human obesity.</a> Br J Nutr. , 1-10 (2010).</li><li>Larsen, M., Bjarkam, C. R., Stoltenberg, M., Sorensen, J. C., Danscher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autometallographic+technique+for+myelin+staining+in+formaldehyde-fixed+tissue.">An autometallographic technique for myelin staining in formaldehyde-fixed tissue.</a> Histol Histopathol. 18, 1125-1130 (2003).</li><li>Alstrup, A. K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+and+Analgesia+in+Ellegaard+G&#246;ttingen+minipigs.">Anaesthesia and Analgesia in Ellegaard G&#246;ttingen minipigs.</a> Jannerup.DK. , (2010).</li><li>Jensen, K. N., Deding, D., Sorensen, J. C., Bjarkam, C. R. <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+implantation+of+deep+brain+stimulation+electrodes+in+the+pontine+micturition+centre+of+the+Gottingen+minipig.">Long-term implantation of deep brain stimulation electrodes in the pontine micturition centre of the Gottingen minipig.</a> Acta Neurochir (Wien). 151, 785-794 (2009).</li><li>Cumming, P., Moller, M., Benda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PET+study+of+effects+of+chronic+3,4-methylenedioxymethamphetamine+(MDMA,+"ecstasy")+on+serotonin+markers+in+Gottingen+minipig+brain.">A PET study of effects of chronic 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") on serotonin markers in Gottingen minipig brain.</a> Synapse. 61, 478-487 (2007).</li><li>Olsen, A. K., Keiding, S., Munk, O. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hypercapnia+on+cerebral+blood+flow+and+blood+volume+in+pigs+studied+by+positron+emission+tomography.">Effect of hypercapnia on cerebral blood flow and blood volume in pigs studied by positron emission tomography.</a> Comp Med. 56, 416-420 (2006).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> J Neurosci Methods. 12, 141-149 (1984).</li></ol>

All procedures were approved by the Danish Experimental Animal Inspectorate, Danish Ministry of Justice and were conducted according to the "Principles of laboratory animal care" (NIH publication No. 86-23, revised 1985).

This presentation provides step-by-step visual instructions on how to perform four frequently used procedures in minipig research12,13. The techniques described may be used with little or no adaptation in landrace pigs and other large research animals such as dogs and sheep14. Aseptic technique must be employed to avoid infections, when performing the described procedures in survival studies. 
Induction of anesthesia is important for animal welfare and scientific reasons, and should provide absence of corneal reflexes and good muscular relaxation, before endotracheal intubation is attempted. However, because the anaesthetics used for induction are not given in fixed doses, but administered until the desired effect is observed, it is important to closely monitor the animal due to the risk of apnoea, especially if using propofol6,10. For tracheal intubation, the use of xylocaine spray or muscle relaxant can facilitate the introduction of the tube, but the use of neuromuscular blockers should only be done by personnel who are well-trained in such procedures so as to conform with ethical requirements. Care should be taken not to traumatize the laryngeal passage during intubation, in order to avoid tracheal swelling and edema. Airway edema can be treated with NSAID or similar anti-inflammatory drugs10. Pigs can be intubated from various positions, but having the animal on its back tends to be easier for those who regularly carry out human intubation, as it presents the airway in a similar manner6. The most common error during intubation is inadvertent placement of the tube in the esophagus, which is situated 
just below the laryngeal passage when the animal is placed on its back. Note, the tube should never be advanced without first clearly identifying the 
arytenoids cartilages and vocal cords.
Transurethral catheterization of the urinary bladder through the urethra is easily done in female pigs, while this procedure is extremely difficult, perhaps impossible, in males6. It should be noted that since the female urethral orifice is anterior to the vagina, the larger vaginal opening can be mistakenly catheterized. The use of sterile and disinfecting lubricant is advisable for survival studies. Furthermore, lubricant with local anesthetic effect may ease the catheterization procedure.
When performing femoral artery and vein catherization, the choice of vessel catheters and how they are secured differ between non-survival and survival studies, especially when catheters are implanted for an extended period of time6. For instance catheters for chronic implantation should be highly biocompatible, have retention beads or cuffs for fixation as well as not traumatize the vessel wall over time, thus inducing thrombosis or erosion6.
In the present video, we use a closed system powered by compressed air for transcardial perfusion. Another option is to use a system driven by gravity. The advantage of gravity systems in comparison to systems utilizing compressed air is that they are simple, efficient, and present no risk of pressurized air entering the vascular system. However, the hydrostatic pressure exerted by gravity systems changes continuously during perfusion, and this can affect the histological results15. Depending on which type of histopathological studies one wants to perform, it may be advantageous to perfuse the animal with heparinized saline first. This is particular importantance when performing immunofluorescent staining in order to avoid autofluorescence problems caused by erythrocytes. Furthermore, if the organ of interest is the brain, the descending aorta could be clamped to save fixative. However, in pigs, this step entails additional dissecting and will prolong the procedure.

<table border="1">
	<tbody>
		<tr>
			<th>Procedure</th>
			<th>Equipment</th>
			<th>Company</th>
			<th>Catalogue-number</th>
		</tr>
		<tr>
			<td>Intubation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Respirator</td>
			<td>Datex-Ohmeda S/S Avance, GE Healthcare</td>
			<td>1009-9002-000</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Suction with blunt tip</td>
			<td>Unomedical</td>
			<td>34091181</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Laryngoscope</td>
			<td>Miller</td>
			<td>85-0045 (Laryngoscope set)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Endotracheal tube 5.5 mm ID</td>
			<td>Smiths Medical International Ltd.</td>
			<td>100/199/055</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Stylet</td>
			<td>Hudson RCI</td>
			<td>5-151001</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Syringe 5ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Stethoscope</td>
			<td>Martin</td>
			<td>17-262-00</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bladder catherization</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>AquaFlate glycerine silicone Foley catheter 8-French with stylet</td>
			<td>R&uuml;sch</td>
			<td>178003</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Speculum nasal</td>
			<td>Martin</td>
			<td>36-859-50</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Speculum rectal</td>
			<td>Martin</td>
			<td>30-613-15</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Blunt-tip forceps</td>
			<td>Martin</td>
			<td>13-374-16</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Catheter lubricant gel</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Cathejell Lidocain</td>
			<td>Montavit</td>
			<td>CJL08501</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Instillagel Comb.</td>
			<td>Farco Pharma</td>
			<td>08-40-200</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>A4 Urine drainage bag</td>
			<td>Unomedical</td>
			<td>38 155 1S</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Adhesive tape</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Femoral artery and vein catherization </</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Scalpel</td>
			<td>Martin</td>
			<td>10-295-11</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Blunt-tip surgical scissors</td>
			<td>Martin</td>
			<td>11-881-18</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Tissue forceps</td>
			<td>Martin</td>
			<td>12-100-14</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Small blunt-tip surgical forceps</td>
			<td>Martin</td>
			<td>13-375-13</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Small self-retaining Tissue retractor</td>
			<td>Martin</td>
			<td>15-753-13</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Needleholder</td>
			<td>Martin</td>
			<td>20-634-14</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Surgical swaps</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Suture with needle</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Saline</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Heparine</td>
			<td>LEO</td>
			<td>585679</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>18-gauge venflon</td>
			<td>BD Venflon Pro</td>
			<td>393206</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>4-French brite tip sheaths with introducer and Seldinger guide wire</td>
			<td>Cordis</td>
			<td>154-2629-1/0017</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transcardial perfusion</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Pressurized perfusion system</td>
			<td>Home made from parts available in most professional hardware stores</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Fume extraction system</td>
			<td>Gram A/S</td>
			<td>01750000 and 0303700</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Perfusant 5L</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Blunt-tip surgical scissors</td>
			<td>Martin</td>
			<td>11-881-18</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Bone shears</td>
			<td>F.S.T</td>
			<td>16150-24</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Large curved tip self-holding forceps</td>
			<td>Martin</td>
			<td>13-421-18</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Self retaining sternal retractor</td>
			<td>Rudolf Medical</td>
			<td>7520-10</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Pentobarbital</td>
			<td>The Royal Veterinary and Agricultural University (KVL)</td>
			<td>200 mg/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>22-gauge venflon</td>
			<td>BD Venflon Pro</td>
			<td>393202</td>
		</tr>
	</tbody>
</table>

basic surgical techniques in the g&ouml;ttingen minipig: intubation, bladder catheterization, femoral vessel catheterization, and transcardial perfusion

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

Watch this Scientific Journal Video about Basic Surgical Techniques in the GÃ¶ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion at JoVE

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

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

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

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

basic-surgical-techniques-gottingen-minipig-intubation-bladder

Research

JoVE Journal

Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

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

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Brachial Artery Catheterization in Swine

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood pressure continuously and collect arterial blood samples to assess arterial blood gas measurements. Arterial blood pressures and arterial blood gases are important physiological parameters to monitor during experimental procedures. In swine, four common methods of arterial catheterization have been described, including catheterization of the carotid, femoral, auricular, and medial saphenous arteries. Each of these techniques have advantages, such as ease of access for the auricular artery, and disadvantages that include deep tissue dissection for carotid artery catheterization. The described alternative method of arterial catheterization in swine, the catheterization of the distal aspect of the brachial artery, is a rapid procedure that requires relatively minimal tissue dissection and provides information that is in line with data collected from other arterial catheterization sites. The procedure uses a medial approach along an oblique plane of the lower brachium, positioned between the olecranon and the flexor aspect of the elbow joint, and this approach allows researchers the major advantage of unimpeded freedom for procedures that involve the caudoventral, caudodorsal back, or hind limbs of the pig. Due to the location of the upper forelimb of the catheterized vessel and potential challenges of effective homeostasis following catheter removal from the artery, this technique may be limited to non-recovery procedures.

The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements.

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood ...