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

Summary

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.

Abstract

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<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.

Introduction

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 consider....

Protocol

All procedures have been approved by the University of Padova Animal Care Committee (OPBA, protocol number n° 55/2017) and authorized by the Italian Ministry of Health (Authorization n° 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

1. 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.
2. Ensure that the weight of the rats ranges from 320-40....

Results

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 .......

Discussion

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.......

Materials

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