The study was funded by the integrated budget for interdepartmental research (BIRD) 2019.

All procedures have been approved by the University of Padova Animal Care Committee (OPBA, protocol number n&#176; 55/2017) and authorized by the Italian Ministry of Health (Authorization n&#176; 700/2018-PR), in compliance with the European Union Directive 2010/63/UE and the Italian Law 26/2014 for the Care and Use of Laboratory Animals.
1. Animal care and experimental model
<ol>
	<li>Ensure all Lewis rats are obtained from a single company (Table of materials). Maintain the rats in conventional facilities with free access to food and water.</li>
	<li>Ensure that the weight of the rats ranges from 320-400 g for the recipient group and 200-250 g for the donor group.</li>
</ol>
2. Preoperative protocol
NOTE: All operations must be performed under clean conditions. Use male and female adult Lewis rats as recipients and donors as well in order to perform a syngeneic transplant.
<ol>
	<li>Perform an intraperitoneal injection of tramadol (5 mg/kg) 15 min before surgery.</li>
	<li>Administer a single dose of intramuscular Gentamicin (5 mg/kg) immediately before surgery.</li>
	<li>For anesthesia induction, supply 4% sevoflurane in 1 L/min of oxygen to a poly(methyl methacrylate) chamber where the animal is placed. For anesthesia maintenance, use 2.0-2.5% sevoflurane in 1 L/min of oxygen throughout the procedure.</li>
	<li>Shave the animal along the midline for 2 cm width from the sternum to 1 cm above the genital area with a razor. Then, sterilize the skin with iodine solution.</li>
	<li>In order to prevent the animal from getting wet and to prevent heat dispersion during the surgery, cover the animal with a transparent plastic film.</li>
	<li>Evaluate the level of anesthesia before performing the procedure by assessing the absence of response to a noxious stimulus.</li>
</ol>
3. Donor operation
<ol>
	<li>Animal and heart preparation:
	<ol>
		<li>Place the anesthetized animal on a cork tray with the caudal side facing the surgeon. Perform a xipho-pubic incision of about 5-6 cm, and retract the two musculocutaneous flaps laterally.</li>
		<li>Administer a volume of 1 mL of saline solution at 4 &#176;C containing 500 IU of heparin through the abdominal vena cava.</li>
		<li>After 1 min, cut the diaphragm from left to right and perform an anterior thoracotomy to expose the heart.</li>
		<li>Cool the beating heart by dripping saline solution at 4 &#176;C.</li>
		<li>Perform a pericardiectomy and a thymectomy in order to obtain a complete view of the aortic arch. Remove the remaining fatty tissues surrounding the aorta.</li>
		<li>Cut at the arch, just above the origin of the innominate artery; sever the latter one, too.</li>
		<li>Cut the thoracic inferior vena cava (IVC) and insert a 22 G cannula to infuse the heart with 20-25 mL of saline solution at 4 &#176;C, exerting light pressure. Discontinue the perfusion when the heart stops beating and flow from the aorta became clear.</li>
	</ol>
	</li>
	<li>PAG explant: 
	NOTE: An accurate harvesting and delicate handling of the PAG is mandatory for achieving optimal implantation in the recipient. Do not touch it directly with instruments, instead use cotton swabs.
	<ol>
		<li>Perform an ultrasound study to assess the PA diameter in its native position.</li>
		<li>Insert a micro-plier under the posterior wall of the vessel and cut the latter using a micro-scissor as close as possible to its bifurcation to maximize the length of the PAG.</li>
		<li>Gently hold the PA with the ring-tipped micro-forceps and separate it from the right ventricle with the micro-spring scissors. Harvest the PAG, including some right ventricular muscle.</li>
	</ol>
	</li>
	<li>PAG preparation:
	<ol>
		<li>Place the PAG on a gauze moistened with cold saline on the operating table and inspect the vessel under the operating microscope.</li>
		<li>Cut any abundant surrounding tissue, leaving only 1 mm of ventricular muscle. Set the length of the vessel at 5 mm.</li>
	</ol>
	</li>
</ol>
4. Pulmonary artery graft (PAG) implantation
<ol>
	<li>Preparation of the recipient animal:
	<ol>
		<li>Place the anesthetized animal on a cork tray with the caudal side facing the surgeon.</li>
		<li>Perform a median longitudinal incision and use two mini retractors to keep the abdomen open.</li>
		<li>Extract the intestines with two cotton swabs and cover them with a gauze soaked with 39 &#176;C saline allowing the visualization of the retroperitoneal area with exposure of the infra-renal abdominal aorta (AA). 
		NOTE: During the surgery, it is important to occasionally moisten the intestines using a syringe containing 39 &#176;C saline to prevent hypothermia, a critical condition common in rodents.</li>
		<li>Strip the posterior parietal peritoneum between the two renal arteries and the iliac bifurcation using two cotton swabs and remove the fatty tissue around the infrarenal AA. Leave only a small portion of fat above the AA, to facilitate handling on the vessel.</li>
		<li>Separate the AA from the IVC. To perform this procedure, first, pass a curved forceps behind the posterior aortic wall and use it to open a passage between the AA and IVC. Then, use a 2-0 silk suture to create a loop around the AA, in order to lift the vessel and separate the AA from IVC. Ligate any lumbar artery arising from the infrarenal AA with 6/0 silk suture and divide it.</li>
		<li>Rotate the animal 90&#176; counterclockwise, placing the head on the operator&#39;s left side. The AA now lay horizontally in the microscopic field.</li>
		<li>Use two Yasargil clips to clamp the infrarenal AA and place them at a distance of 1.5 cm from each other. Transect the AA at the midpoint between the two clips.</li>
		<li>Irrigate the two ends of the vessels with heparin (1 UI/mL) in saline solution to remove any clots. Remove any adventitial debris from the vessels.</li>
	</ol>
	</li>
	<li>PAG implantation:
	<ol>
		<li>Place the PAG between the two ends, with the ventricular end towards the cranial portion of the animal.</li>
		<li>Use a 10-0 polypropylene suture to perform two landmark single stitches connecting the PG to the AA. Perform the procedure on both ends of the PAG by placing the suture on opposite sides of the vessel circumference.</li>
		<li>Perform an end-to-end anastomosis between PAG and AA, beginning with the distal end. Use one of the two ends of the distal landmark suture for the posterior anastomosis using a recipient-to-graft out-in/in-out sequence to perform a running suture of about six stitches.</li>
		<li>Once the suture reaches the proximal landmark, perform a double half hitch completed by a square knot using the suture and one of the two ends of the proximal landmark suture. Apply rubber-shod mosquito forceps to the sutures to provide traction.</li>
		<li>Perform the same anastomosis on the anterior wall. Carry on the entire procedure on the proximal end of the PAG. Take particular attention when performing the proximal anastomosis to avoid including any leaflet in the suture line.</li>
		<li>Release the distal clip first to let the PAG be filled with retrograde blood (low-pressure flow) in order to check the anastomosis. Repair any blood leakage with a single suture. Once the distal anastomosis is evaluated, perform the same procedure on the proximal end.</li>
	</ol>
	</li>
	<li>Final stages of the operation on the recipient:
	<ol>
		<li>Assess the patency of the PAG and apply two strips of gelatin sponge over the suture lines on both sides of the PAG (if necessary). Exert gentle pressure for a few seconds with two cotton swabs to help hemostasis.</li>
		<li>Relocate the intestines into the abdominal cavity and close the walls with a 4/0 polypropylene running suture.</li>
	</ol>
	</li>
</ol>
5. Sham-operated procedure
<ol>
	<li>Perform an identical preparation of the animal as previously illustrated for recipient rats.</li>
	<li>Cut the infra-renal AA, midway between the renal and the iliac arteries&#39; origin.</li>
	<li>Reapproximate the two ends of the AA using an end-to-end anastomosis, as previously described. Remove the two clips and perform an accurate hemostasis procedure.</li>
	<li>Reposition the intestines and close the abdominal wall in layers, as for the recipient animals.</li>
</ol>
6. Postoperative care and follow-up
<ol>
	<li>Administer warm saline solution (5 mL) into the subcutaneous tissue of the animal&#39;s back for hydration. Place the rat under a heating lamp and visually monitor it until awakening, which usually takes up to 5 min after anesthesia is stopped. Place the animal in a cage at a room temperature of 22-24 &#176;C, with immediate and unrestricted access to food and water.</li>
	<li>Administer intramuscular tramadol (5 mg/kg) for postoperative analgesia twice daily for the first 48 h after surgery. Thereafter, monitor the recipient&#39;s health status and body weight daily, on a regular basis.</li>
	<li>Follow-up: During the follow-up, perform seriate ultrasound studies at one week, one month, and two months to evaluate PAG function. During these studies, measure the vessel diameter, the peak systolic velocity (PSV), and the end-diastolic velocity. Measure these parameters inside the PAG and at the level of proximal and distal AA.</li>
	<li>Euthanize the animals after two months of follow-up by application of CO2 for a few minutes, and then explant the PAG, which will undergo histopathological analysis.</li>
</ol>

<ol>
	<li>Botto, L. D., Correa, A., Erickson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+and+temporal+variations+in+the+prevalence+of+heart+defects.">Racial and temporal variations in the prevalence of heart defects.</a> Pediatrics. 107 (3), 32 (2001).</li><li>Vergnat, M., 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=Aortic+stenosis+of+the+neonate:+A+single-center+experience.">Aortic stenosis of the neonate: A single-center experience.</a> The Journal of Thoracic and Cardiovascular Surgery. 157 (1), 318-326 (2019).</li><li>Hra&#353;ka, 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=The+long-term+outcome+of+open+valvotomy+for+critical+aortic+stenosis+in+neonates.">The long-term outcome of open valvotomy for critical aortic stenosis in neonates.</a> The Annals of Thoracic Surgery. 94 (5), 1519-1526 (2012).</li><li>Kaza, A. K., Pigula, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+bioprosthetic+valves+appropriate+for+aortic+valve+replacement+in+young+patients.">Are bioprosthetic valves appropriate for aortic valve replacement in young patients.</a> Seminars in Thoracic and Cardiovascular Surgery: Pediatric Cardiac Surgery Annual. 19 (1), 63-67 (2016).</li><li>Myers, P. O., 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=Outcomes+after+mechanical+aortic+valve+replacement+in+children+and+young+adults+with+congenital+heart+disease.">Outcomes after mechanical aortic valve replacement in children and young adults with congenital heart disease.</a> The Journal of Thoracic and Cardiovascular Surgery. 157 (1), 329-340 (2019).</li><li>Donald, J. S., 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=Ross+operation+in+children:+23-year+experience+from+a+single+institution.">Ross operation in children: 23-year experience from a single institution.</a> The Annals of thoracic surgery. 109 (4), 1251-1259 (2020).</li><li>Khwaja, S., Nigro, J. J., Starnes, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ross+procedure+is+an+ideal+aortic+valve+replacement+operation+for+the+teen+patient.">The Ross procedure is an ideal aortic valve replacement operation for the teen patient.</a> Seminars in Thoracic and Cardiovascular Surgery: Pediatric Cardiac Surgery Annual. , 173-175 (2005).</li><li>Elkins, R. C., Lane, M. M., McCue, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ross+operation+in+children:+late+results.">Ross operation in children: late results.</a> The Journal of Heart Valve Disease. 10 (6), 736-741 (2001).</li><li>Chambers, J. C., Somerville, J., Stone, S., Ross, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+autograft+procedure+for+aortic+valve+disease:+long-term+results+of+the+pioneer+series.">Pulmonary autograft procedure for aortic valve disease: long-term results of the pioneer series.</a> Circulation. 96 (7), 2206-2214 (1997).</li><li>Mazine, 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=Ross+procedure+in+adults+for+cardiologists+and+cardiac+surgeons:+JACC+state-of-the-art+review.">Ross procedure in adults for cardiologists and cardiac surgeons: JACC state-of-the-art review.</a> Journal of the American College of Cardiology. 72 (22), 2761-2777 (2018).</li><li>Sengupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+rat:+Relating+its+age+with+humans.">The laboratory rat: Relating its age with humans.</a> International Journal of Preventive Medicine. 4 (6), 624-630 (2013).</li><li>Ashfaq, A., Leeds, H., Shen, I., Muralidaran, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinforced+ross+operation+and+intermediate+to+long+term+follow+up.">Reinforced ross operation and intermediate to long term follow up.</a> Journal of Thoracic Disease. 12 (3), 1219-1223 (2020).</li><li>Vida, V. L., 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=Age+is+a+risk+factor+for+maladaptive+changes+of+the+pulmonary+root+in+rats+exposed+to+increased+pressure+loading.">Age is a risk factor for maladaptive changes of the pulmonary root in rats exposed to increased pressure loading.</a> Cardiovascular Pathology: The Official Journal of the Society for Cardiovascular Pathology. 21 (3), 199-205 (2012).</li><li>Nappi, F., 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=An+experimental+model+of+the+Ross+operation:+Development+of+resorbable+reinforcements+for+pulmonary+autografts.">An experimental model of the Ross operation: Development of resorbable reinforcements for pulmonary autografts.</a> The Journal of Thoracic and Cardiovascular Surgery. 149 (4), 1134-1142 (2015).</li><li>Vanderveken, E., 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=Mechano-biological+adaptation+of+the+pulmonary+artery+exposed+to+systemic+conditions.">Mechano-biological adaptation of the pulmonary artery exposed to systemic conditions.</a> Scientific Reports. 10 (1), 2724 (2020).</li></ol>

The authors have nothing to disclose.

Aortic valve replacement with the autologous pulmonary root (Ross operation) represents an attractive option for congenital aortic valve stenosis repair due to the favorable profile and potential growth of the autograft10. The major limitation to this procedure is the potential dilatation of the aortic neo-valve, which predisposes to the development of long-term regurgitation. The possibility of characterizing the modifications on the pulmonary artery after exposure to systemic pressures could rep...

Congenital aortic valve stenosis is a subgroup of congenital heart disease characterized by an obstruction of the left ventricular tract in which the lesion is located at the valvular level. The malformation affects approximately 0.04-0.38 per 1000 live births1.
The available options for the correction are many, each with its own advantages and disadvantages. For patients suitable for a biventricular correction2, the approach may be aimed at valve repair (percutaneous or surgical valvulotomy) or its replacement3. The latter is preferred when the aortic valve is considered unsalvageable; however, the available options are limited for pediatric patients. Indeed, bioprosthetic valves are not indicated for aortic replacement in the young population due to their early calcification4. On the other hand, degeneration in mechanical valves is considerably slower, but these require lifelong anticoagulant therapy5. In addition, the major limitation of these prostheses is represented by the lack of growth potential, which predisposes the patients to additional reinterventions.
An interesting therapeutic option in the pediatric population is the transfer of the pulmonary autograft to the aortic position named &#34;Ross operation&#34;. In this case, the pulmonary valve is then replaced with a homograft (Figure 1)6. This procedure can possibly represent the best surgical choice for children because the pulmonary autograft preserves its growth potential and does not carry the risks of lifelong anticoagulant therapy. Furthermore, the Ross procedure can be of great value also in young adults to avoid a mechanical or biological valve, having the potential to become the best surgical solution.
Results after aortic valve replacement with pulmonary autograft are excellent, with survival greater than 98% and good long-term outcomes7. Literature studies report 93% and 90% freedom from replacing the pulmonary homograft at 4 and 12 years, respectively8.
The major limitation of this procedure is the tendency of the autograft to dilate in the long term, especially when employed as a freestanding root replacement. This can cause valvular incompetence which may require a reintervention. Indeed, the longest follow-up study performed so far reports freedom from reoperation for autograft replacement of 88% at 10 years and 75% at 20 years9.
The possibility of recreating a Ross operation in an experimental setting represents a fundamental prerequisite to investigate the underlying mechanism of the pulmonary autograft adaption to systemic pressures. Several models have been proposed in the past. However, these are usually limited to ex-vivo experiments or in-vivo animal models with a relatively expensive large animals. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position, as freestanding root.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Monico SpA</td>
			<td>AIC 030805105</td>
			<td>Two bottles of 100 mL. The cold one (4&#176;C) for flushing the harvesting organ; the warm one (39&#176;C) for moistening, and rehydration of the recipient</td>
		</tr>
		<tr>
			<td>7.5% Povidone-Iodine</td>
			<td>B Braun</td>
			<td>AIC 032151211</td>
			<td></td>
		</tr>
		<tr>
			<td>Barraquer</td>
			<td>Aesculap</td>
			<td>FD 232R</td>
			<td>Straight micro needle holder for the vascular anastomoses</td>
		</tr>
		<tr>
			<td>Castroviejo needle holder</td>
			<td>Not available</td>
			<td>J 4065</td>
			<td>To close the animal</td>
		</tr>
		<tr>
			<td>Clip applying forceps</td>
			<td>Rudolf Medical</td>
			<td>RU 3994-05</td>
			<td>For clip application</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Johnson &#38; Johnson Medical SpA</td>
			<td>N/A</td>
			<td>Supermarket product. Sterilized</td>
		</tr>
		<tr>
			<td>Curved micro jeweller forceps</td>
			<td>Rudolf Medical</td>
			<td>RU 4240-06</td>
			<td>Used to pass sutures underneath the vases.</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>RB healthcare</td>
			<td>N/A</td>
			<td>Supermarket product</td>
		</tr>
		<tr>
			<td>Electrocautery machine</td>
			<td>LED SpA</td>
			<td>Surton 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>Rudolf Medical</td>
			<td>RU 2422-11</td>
			<td>For opening the abdomen (recipient)</td>
		</tr>
		<tr>
			<td>Fine-tip curved Vannas micro scissors</td>
			<td>Aesculap</td>
			<td>OC 497R</td>
			<td>Only for preparing the pulmonary root, cut the lumbar vases and the 10/0 Prolene</td>
		</tr>
		<tr>
			<td>Fluovac Isoflurane/Halotane Scavanger unit</td>
			<td>Harvard Apparatus Ltd</td>
			<td>K 017041</td>
			<td>Complete of anesthesia machine, anesthesia tubing, induction chamber and scavenger unit with absorbable filter</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>MSD Italia Srl</td>
			<td>AIC 020891014</td>
			<td>Antibiotic. Single dose, 5 mg/kg intramuscular, administered during surgery</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Pharmatex Italia Srl</td>
			<td>AIC 034692044</td>
			<td>500 IU into the recipient abdominal vena cava</td>
		</tr>
		<tr>
			<td>I.V. Catheter</td>
			<td>Smiths Medical Ltd</td>
			<td>4036</td>
			<td>20G</td>
		</tr>
		<tr>
			<td>Insulin Syringe, 1 mL</td>
			<td>Fisher Scientific</td>
			<td>14-841-33</td>
			<td>To inject heparin in the harvesting animal and to flush the sectioned aorta in the recipient</td>
		</tr>
		<tr>
			<td>Jeweler bipolar forceps</td>
			<td>GIMA SpA</td>
			<td>30665</td>
			<td>0.25 mm tip. For electrocautery of very small vases</td>
		</tr>
		<tr>
			<td>Lewis rats (LEW/HanHsd)</td>
			<td>Envigo RMS SRL, San Pietro al Natisone, Udine, Italy</td>
			<td>86104M</td>
			<td>Male or female, weighing 200-250 g (pulmonary root harvesting animals) and 320-400 g (recipients)</td>
		</tr>
		<tr>
			<td>Micro-Mosquito</td>
			<td>Rudolf Medical</td>
			<td>RU 3121-10</td>
			<td>In number of four, with tips covered with silicon tubing. To keep in traction the Prolene suture during anastomosis</td>
		</tr>
		<tr>
			<td>Operating microscope</td>
			<td>Leica Microsystems</td>
			<td>M 400-E</td>
			<td>Used with 6x, 10x and 16x in-procedure interchangeable magnifications</td>
		</tr>
		<tr>
			<td>Perma-Hand silk 2-0</td>
			<td>Johnson &#38; Johnson Medical SpA</td>
			<td>C026D</td>
			<td>To lift the aorta</td>
		</tr>
		<tr>
			<td>Petrolatum ophthalmic ointment</td>
			<td>Dechra</td>
			<td>NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolene 10-0</td>
			<td>Johnson &#38; Johnson Medical SpA</td>
			<td>W2790</td>
			<td>Very fine non-absorbable suture, with a BV75-3 round bodied needle, for the vascular anastomoses</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Not any</td>
			<td>N/A</td>
			<td>Two home-made retractors</td>
		</tr>
		<tr>
			<td>Ring tip micro forceps</td>
			<td>Rudolf Medical</td>
			<td>RU 4079-14</td>
			<td>For delicate manipulation</td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>AbbVie Srl</td>
			<td>AIC 031841036</td>
			<td>Mixed with oxygen, for inhalatory anesthesia</td>
		</tr>
		<tr>
			<td>Spring type micro scissors</td>
			<td>Rudolf Medical</td>
			<td>RU 2380-14</td>
			<td>Straight; 14 cm long</td>
		</tr>
		<tr>
			<td>Standard aneurysm clips</td>
			<td>Rudolf Medical</td>
			<td>RU 3980-12</td>
			<td>Two clips (7.5 mm; 180 g; 1.77 N) to close the aorta</td>
		</tr>
		<tr>
			<td>Sterile gauze of non-woven fabric material</td>
			<td>Luigi Salvadori SpA</td>
			<td>26161V</td>
			<td>7.5x7.5 cm, four layers</td>
		</tr>
		<tr>
			<td>Straight Doyen scissors</td>
			<td>Rudolf Medical</td>
			<td>RU/1428-16</td>
			<td>For use to the donor</td>
		</tr>
		<tr>
			<td>Straight micro jeweller forceps</td>
			<td>Rudolf Medical</td>
			<td>RU 4240-04</td>
			<td>10.5 cm long. Used throughout the anastomosis</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Artsana SpA</td>
			<td>N/A</td>
			<td>20 mL (for the harvesting animal) and 5 mL (for the recipient). For saline flushing and dipping</td>
		</tr>
		<tr>
			<td>TiCron 4-0</td>
			<td>Covidien</td>
			<td>CV-331</td>
			<td>For closing muscles and skin</td>
		</tr>
		<tr>
			<td>Tissue forceps V. Mueller</td>
			<td>McKesson</td>
			<td>CH 6950-009</td>
			<td>Used for skin and muscles</td>
		</tr>
		<tr>
			<td>Tramadol</td>
			<td>SALF SpA</td>
			<td>AIC 044718029</td>
			<td>Analgesic. Single dose, 5 mg/kg intramuscular</td>
		</tr>
		<tr>
			<td>Virgin silk 8-0</td>
			<td>Johnson &#38; Johnson Medical SpA</td>
			<td>W818</td>
			<td>For arterial branch ligation</td>
		</tr>
	</tbody>
</table>

a rodent model of the ross operation: syngeneic pulmonary artery graft implantation in a systemic position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

A total of 39 adult Lewis rats were included in this study: 17 animals were used as PAG donors, 17 animals as recipients and 5 as sham-operated (control group) (Table 1). Male rats were 22 (56%) and female 17 (44%); the latter were used only in the donor group.
No fatal event occurred during the operation with 100% survival. During the follow-up, two animals of the transplant group had a fatal outcome, at 12 and 51 days, respectively; the survival rate at the end of the study ...

Watch this Scientific Journal Video about A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position at JoVE.com

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...

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3.0K Views.  University of Padua. We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

Video: A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...