Name: a rodent model of the ross operation: syngeneic pulmonary artery graft implantation in a systemic position
Uploaded: 2022-04-01T14:00:00
Description: 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

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</stro...

<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>

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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