Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Summary

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Abstract

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient’s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

Introduction

Congenital cardiovascular defects are one of the leading causes of infant mortality in the western world^1,2.  Among them, pulmonic valve stenosis and bicuspid aortic valve defects are a frequently occurring form³.  Heart valve replacement surgery is a routine choice of reconstructive surgeries; however, complications including stenosis and calcification of the heart valve, and lifelong dependence on anticoagulants are a significant source of chronic ill health and death^4-7. Moreover, the lack of growth potential requires revision surgeries, which further increases the mortality of those young patients^4,8,9.

In an attempt to develop a functional replacement heart valve with growth potential, Shinoka et al. seeded autologous cells onto a biodegradable synthetic heart valve⁸. The synthetic valve transformed to a native heart valve like structure with growth potential. Preliminary large animal studies demonstrated the feasibility of using this methodology to create a functional heart valve¹⁰.  However, long term implantation studies demonstrated poor durability due to progressive thickening of the valve neotissue resulting in narrowing of the heart valve. Work from Sodian et al. used the Shinoka methodology, but ultimately replaced the PGA matrix with a biodegradable elastomer, which gave the biomechanical properties of the tissue engineered valve construct a more physiological profile^9,11,12. In the in vivo study, despite the success of the implantation, a confluent endothelial cell lining was not formed which could limit the long term success of this scaffold¹².

In order to rationally design an improved second generation synthetic heart valve, a murine model of heart valve transplantation was created to investigate the cellular and molecular mechanisms underlying neotissue formation, valve thickening, and stenosis development. Murine models offer a vast array of molecular reagents, including transgenics, which are not readily available in other species⁷. In this heart valve transplantation model, an ex vivo syngeneic pulmonary heart valve replacement was performed first; and then the heart with the implanted heart valve was implanted heterotopically into a syngeneic host using a microsurgical technique. This model enables heart valve replacement without the need for cardiopulmonary bypass.

In this paper, a detailed explanation of a heart valve harvest, donor heart preparations, heart valve transplantation, and heterotopic heart transplantation is described. The results showed a continuous heartbeat from the donor heart, which was independent of the recipient heartbeat. The blood flow through the implanted pulmonary valve was measured using a high frequency ultrasound system with a pulsed wave Doppler.

Protocol

Note: All animal procedures were approved by the Nationwide Children’s Hospital Institutional Animal Care and Use Committee.

1. Pulmonary Heart Valve Harvest from a Heart Valve Donor Mouse

1. Autoclave all the surgical tools before the surgery: 1x fine scissors, 3x micro forceps, 2x micro vascular clamps, 1x clamp applying forceps, 1x micro needle holder, 1x spring scissors, 1x retractor.
2. A 6-8 week old female C57BL/6 mouse is used as a pulmonary heart valve donor. Remove the mouse from its cage and weigh it, then euthanize with a ketamine/xylazine cocktail (Ketamine, 200 mg/kg and xylazine, 20 mg/kg, IP) overdose.
3. Clip the chest area and place the mouse in a dorsal recumbence position on a pad. Then make the thoracotomy. Expose the heart, make a small cut on the right atrium, and perfuse the left ventricle with ice cold saline.
4. Bluntly dissect the pulmonary artery (PA) from the ascending aorta. Cut out the pulmonary valve (PV) along with 2 mm cuff of pulmonary artery. Dispose of the remainder of the heart.
5. Store the PV in cold heparin and saline solution (100 units/ml). Note: The PV can be kept in the solution for two hours before transplantation to the donor heart.

2. Donor Heart Preparation

1. A 6-8 week old female C57BL/6 mouse is used as a heart donor. Remove the mouse from its cage and weigh it, then euthanize with a ketamine/xylazine cocktail (ketamine, 200 mg/kg and xylazine, 20 mg/kg, IP) overdose. This is a terminal procedure. 
2. Clip the chest area and place the mouse in a dorsal recumbence position on a pad. Then make the thoracotomy. Bluntly separate the heart, inferior vena cava (IVC), superior vena cava (SVC), ascending aorta, PA, and pulmonary vein. Perfuse the IVC with ice cold sterile saline.
3. Ligate the IVC, SVC, and pulmonary vein with 6-0 silk suture then cut superior to the ligatures.
4. Cut the aorta and PA with 2 mm cuff.
5. Cut out the PV and dispose of it.

3. Heart Valve Transplantation onto a Donor Heart

1. Immediately after step 2.5, place the heart valve from step 1.5 into the donor heart and orient the heart valve.
2. Secure the PV with a stitch on the right side of the valve using 10-0 monofilament sutures on tapered needles and start to suture continuously with 5-6 stiches from the other side of the PV.
3. After finishing the front side, rotate the heart horizontally and start to suture the back side of the PV onto the donor heart.
4. Store the heart in a cold sterile heparin/saline solution. Note: The donor heart can be kept in the solution for two hours before implantation to the recipient mouse.

4. Heterotopic Heart Transplantation on to a Recipient Mouse

1. A 6-8 week old female C57BL/6 mouse was used as a recipient. Remove the mouse from its cage and weigh it, then anesthetized with ketamine/xylazine cocktail (ketamine, 100 mg/kg and xylazine 10 mg/kg). Ketoprofen (5 mg/kg) is used as preanesthesia analgesic.
2. After checking the level of sedation by tail pinching, clip the abdominal and chest hair. Lubricate the eyes with sterile ophthalmic ointment, and place the mouse in a dorsal recumbence position on a pad. Disinfect the abdomen with betadine and alcohol pads. Then cover the mouse with a sterile drape and expose the incision area only.
3. Make a midline laparotomy incision from below the xyphoid to the suprapubic region, and insert a self retaining retractor. Wrap the intestines in saline moistened gauze. Bluntly define the infrarenal aorta and vena cava.
4. Place two 6-0 silk sutures proximally and distally around the aorta and IVC to restrain blood circulation.
5. Place the donor heart on the right side of the abdominal aorta and cover it with sterile gauze. Moisturize it with saline.
6. Make an aortotomy in the abdominal aorta using a 30 G needle and extend the opening with scissors to the size of the donor aorta.
7. Perform an end-to side anastomosis using sterile 10-0 monofilament sutures on tapered needles. Secure the donor aorta with one stitch on proximal end of the opening in the abdominal aorta and start to suture continuously with 4-5 stiches from the distal end of the abdominal aorta.
8. Flip the heart to the left side, cover it with saline infused gauze, and start to suture continuously with 4-5 stiches from the distal end of the abdominal aorta.
9. Make a venotomy in the IVC using a 30 G needle and extend the opening to the size of the donor pulmonary artery.
10. Perform an end to side anastomosis using sterile 10-0 monofilament sutures on tapered needles. Secure the donor PA with one stitch on the proximal end of the opening in the IVC and start to suture continuously with 4-5 stiches from the distal end of the inferior vena cava. This time, because the aorta is in the way, make sure suturing of the left wall of the donor’s PA is on the inside of the IVC.
11. Flush the IVC lumen with heparin and saline solution (100 units/ml). Close the right wall of the donor PA and recipient IVC by suturing them continuously to the distal end.
12. Remove the distal ligature and control the hemorrhage by applying a topical absorbable sterile hemostat agent. When the hemorrhage stops completely, remove the proximal suture and control the hemorrhage the same way.
13. Return the intestines and close the abdominal musculature and skin in two layers using a 6-0 black polyamide monofilament suture.
14. Inject 0.5 ml saline subcutaneously and place the mouse in a recovery cage on a warming pad until the mouse is fully mobile. Upon recovery, return the mouse to a new cage with paper bedding. Give pain medication (Ibuprofen, 30 mg/kg, drinking water) for 48 hr. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.

Results

Figure 1 illustrates the schematics of the heart valve transplantation model using heterotopic heart transplantation. The heart valve was harvested from a donor heart and implanted onto a heart from a second donor mouse. Then the heart with the new heart valve was implanted to the abdomen of a recipient mouse. Figure 2 shows an illustration of the implanted heart on the abdominal space (A), right after heart transplantation (B), and 5 min after transplan...

Discussion

The mortality rate of this procedure is close to 20%, which was mostly caused by hemorrhage at the PV transplantation site and anastomosis on the donor aorta to the recipient abdominal aorta. In most of the cases, the mortality rate decreases significantly 48 hr post surgery. The survival mice showed strong heart beats and blood flow through the implanted PV. The entire process takes four hours for an experienced micro surgeon. It will take roughly 250 mice to master the technique. The heterotopic heart transplantation i...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DPBS	Gibco	14190-144
Microscope	Leica	M80
C57BL/6J (H-2b), Female	Jackson Laboratories	664	8-12 weeks
Ketamine Hydrochloride Injection	Hospira Inc.	NDC 0409-2053
Xylazine Sterile Solution	Akorn Inc.	NADA# 139-236
Ketoprofen	Fort Dodge Animal Health	NDC 0856-4396-01
Ibuprofen	PrecisionDose	NDC 68094-494-59
Heparin Sodium	Sagent Pharmaceticals	NDC 25021-400
Saline solution (Sterile 0.9% sodium chloride)	Hospira Inc.	NDC 0409-0138-22
0.9% Sodium chloride Injection	Hospira Inc.	NDC 0409-4888-10
Petrolatum Ophthalmic Ointment	Dechra Veterinary Products	NDC 17033-211-38
Iodine Prep Pads	Triad Disposables, Inc.	NDC 50730-3201-1
Alcohol Prep Pads	McKesson Corp.	NDC 68599-5805-1
Cotton tipped applicators	Fisher Sientific	23-400-118
Fine Scissor	FST	14028-10
Micro-Adson Forcep	FST	11018-12
Clamp Applying Forcep	FST	00072-14
S&T Vascular Clamp	FST	00396-01
Spring Scissors	FST	15008-08
Colibri Retractors	FST	17000-04
Dumont #5 Forcep	FST	11251-20
Dumont #7 - Fine Forceps	FST	11274-20
Dumont #5/45 Forceps	FST	11251-35
Tish Needle Holder/Forceps	Micrins	MI1540
Black Polyamide Monofilament Suture, 10-0	AROSurgical Instruments Corporation	TI638402	For sutureing the graft
Black Polyamide Monofilament Suture, 6-0	AROSurgical Instruments	SN-1956	For musculature and skin closure
Non Woven Sponges	McKesson Corp.	94442000
Absorbable hemostat	Ethicon	1961
1 ml Syringe	BD	309659
3 ml Syringe	BD	309657
10 ml Syringe	BD	309604
18 G 1 1/2 in, Needle	BD	305190
25 G 1 in., Needle	BD	305125
30 G 1 in., Needle	BD	305106
Warm Water Recircultor	Gaymar	TP-700
Warming Pad	Gaymar	TP-22G
Trimmer	Wahl	9854-500
VEVO2100 High Frequency Ultrasound	VisualSonics	http://www.visualsonics.com/vevo2100	The catalog number and pricing can be acquired from the sales representatives.
Ultrasound transmission gel	Parker Laboratories, INC.	01-02
Table Top Laboratory Animal Anesthesia System	VetEquip, INC.	901806
Isoflurane	Baxter	1001936060