A Modified Method for Heterotopic Mouse Heart Transplantion

Summary

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Abstract

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37 °C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

Introduction

Animal models of organ transplantation can provide valuable information for improving treatment of clinical transplant patients. Mouse models are particularly useful for studying the immune mechanism of organ transplant rejection or acceptance due to the wide range of genetically modified mice and reagents specific for mice that are not available for other animal models^1-3. A challenge with mouse models of transplantation is the small size of the donors and recipients which requires considerable technical skill to achieve satisfactory outcomes.

A technique was first described for heterotopic transplantation of hearts in rats^....

Protocol

Prior to commencement of experiments, obtain approval from the relevant institution’s Animal Care Ethics Committee for the planned experiments. Maintain mice in the accordance with the requirements of your institution. The following protocol has been approved by Sydney University and Royal Prince Alfred Hospital committees.

Prior to commencing surgery, all instruments should be cleaned thoroughly and sterilized by soaking in 80% ethanol. Some institutions stipulate autoclaving however this can damage fine microsurgical instruments over the long term.  Where possible use sterile disposable equipment.

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Results

After an initial training period, 227 cases of mouse heterotopic heart transplantation in our group were analyzed. The success rate within the first 24 hr was 90.3% and at 48 hr it was 86.8%. Of the 30 (13.2%) failures within 48 hr, 5 (2.2%) experienced hind limb paralysis and had to be euthanized, 10 (4.4%) had a non-beating heart due to graft ischemia injury and/or thrombosis, while 15 (6.6%) died within 48 hr. Some experimental graft survivals are shown in Table 1 with a variety of strain combinations.......

Discussion

Mouse heart transplantation is a challenging microsurgical method that requires considerable surgical skill to master. The most challenging aspect is the small diameter of the vessels. In addition, it is necessary to limit the recipient operation time and bleeding. The technique for mouse heart transplantation was first described by Corry et al. in 1973 and subsequently by Mottram et al¹². Our modifications include the following points. Firstly, immediate perfusion of the donor heart with col.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Operating microscope	Leica, Heerbrugg, Switzerland	M651	10-25X magnification
Anesthetic machine	Vet Quip Pty Ltd, Sydney, Australia	Vett3	Capable of delivering a mixture of isoflurane and oxygen in air
Operating board	Hardware store or office supplier		Dense cork or synthetic capable of taking pins
Heparinized saline (5 U/ml, 4 °C)	Pfizer, USA	FW25	In 1 ml syringe with 23 G needle on ice
Normal saline (37 °C)	AstraZeneca Pty Ltd, Australia	4538	In 1 ml syringe with 23 G needle on warm pad
Sutures 10- nylon, 5-0 Vicryl	Ethicon, Inc. NJ, USA	2870G/J421H
Buprenorphine (0.05 mg/kg in 0.1 ml saline)	Reckitt Benckiser, Sydney, Australia		In 1 ml syringe with 23 G needle on ice
Ampcillin (5 mg/kg in 0.1 ml saline)	Aspen, Sydney, Australia		In 1 ml syringe with 23 G needle on ice
Gel Foam	Pharmacia & Upjohn Co. USA	801289304	Cut into small pieces
High temperature cautery device	Medtronic, USA	8444000
Heating Pad/Right Temp	Kent Scientific Corporation, Turrington, CT 06790
Microsurgery instruments:	Shanghai Medical Instruments Co. Ltd.,
Microneedleholders	Shanghai, China	WT2020
Microscissors	"          "	WT1020
Microforceps (straight tip)	"          "	WA3010
Microforceps (curved tip)	"          "	WA3020
Micromosquito clamps (1 pair)	"          "	W40350
Microvessel atraumatic clamps (1 pair)	"          "	W40130/W40150