Cuff-Free Suture-Based Cervical Model for Secondary Heterotopic Heart Transplantation to Evaluate Donor-Specific Immune Tolerance in Mice

Summary

This protocol presents a cuff-free, suture-based cervical heterotopic heart transplantation technique in mice, designed to enable the reliable placement of a second heterotopic cardiac graft for evaluating donor-specific immune tolerance under physiological conditions with minimal interference from foreign material.

Abstract

Murine heterotopic cardiac transplantation is a well-established model for investigating allograft rejection and immune tolerance. In tolerance studies, the placement of a second heterotopic cardiac graft is often required to assess donor specificity, with the cervical region providing an ideal anatomical site. While cuff-based techniques simplify vascular anastomoses, these methods introduce artificial materials that can promote peri-anastomotic inflammation, alter hemodynamics, and confound immunological analyses. Additionally, vessel narrowing resulting from genetic modifications, immunosuppressive treatment, advanced recipient age, or chronic experimental conditions can make cuff placement technically challenging in small-caliber vessels. Here, we present a cuff-free, suture-based technique for cervical heterotopic heart transplantation in mice. This method avoids the use of intraluminal polymers, preserves physiological blood flow, and reduces complications associated with altered flow dynamics. The protocol includes meticulous preparation of both the donor and recipient vessels, end-to-end micro-anastomoses of the carotid artery and external jugular vein, intraoperative patency assessments, and postoperative monitoring strategies. By minimizing the use of artificial materials and adapting to small-caliber vessels, this cuff-free technique establishes a physiological and reproducible platform for secondary heart transplantation. This approach enables rigorous mechanistic studies of donor-specific tolerance in murine cardiac transplantation models.

Introduction

Heart transplantation is a life-saving intervention for patients with end-stage heart failure¹. Recipients with cardiac allografts require lifelong immunosuppressive therapy to prevent rejection². Despite immunosuppression, long-term graft survival remains compromised by acute and chronic rejection, particularly antibody-mediated rejection (AMR) and cardiac allograft vasculopathy (CAV)³. In addition, these regimens carry significant side effects, including nephrotoxicity, metabolic syndrome, infections, and malignancies, which reduce both graft and patient survival^2,

Protocol

Mice were purchased from Jackson Laboratory. Both male and female mice, aged 6 - 10 weeks and weighing 20 - 30 g at the time of the first abdominal cardiac transplant, were included in this study. Donors and recipients were housed under specific pathogen-free (SPF) conditions at the rodent facility of Massachusetts General Hospital. All procedures were performed humanely in accordance with the NIH guidelines for the care and use of laboratory animals. All animal experiments were approved by and conducted under the oversight of the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (protocol number 2020N000125). The Table of M....

Results

A schematic overview of the cuff-free cervical heterotopic heart transplantation procedure is shown in Figure 1. The microscopic view of the vascular anastomoses is shown in Figure 1A, where the donor ascending aorta is anastomosed end-to-end to the recipient carotid artery and the donor pulmonary trunk is anastomosed end-to-end to the recipient external jugular vein. Figure 1B demonstrates the blood flow pattern through the graft. .......

Discussion

Murine heart transplantation models have played a critical role in advancing our understanding of transplant immunology. Since the development of the original heterotopic abdominal model, these systems have been utilized to characterize the cellular and molecular mechanisms of rejection, test novel immunosuppressive regimens, and evaluate genetic modifications in both donor and recipient strain^8,17,31. The ability to use inbred .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5ml insulinsyringe	Covidien	8881600350	Subcutaneously injection
0.9% Sodium Chloride Injection	BD	1727170103	Intraveneous injection
10-0 nylon suture	AROSurgical Instruments	T04A10N07-13	For mice microscopic surgery
11-0 nylon suture	ETHICON	9016G	For mice microscopic surgery
1ml Syringe	BD	309659	Intraveneous injection
30G X 1/2 Needle	BD	305106	Intraveneous injection
3ml Syringe	BD	309657	Intraveneous injection
6-0 nylon suture	ETHICON	J212H	For mice microscopic surgery
60mm cell culture plate	FALCON	353002	For graft preservation
Colored Aluminum Cases	Fine Science Tools	20330-04	For mice microscopic surgery instruments disinfection
Cotton Tips	Fisherbrand	14-960-3M	Stop bleeding
Dumont #5 Forceps	Fine Science Tools	11251-20	For mice microscopic surgery
Ethiqa XR Buprofenor (CIII) VI	RX GENERICS	86084010030	Analgesia
Fine sissors	Fine Science Tools	15400-12	For mice microscopic surgery
Gauze Sponges	MEDLINE	NON25334	Protect the graft and interstine
Heat Lamp with Base	Braintree Scientfic	HL-1B	Post surgery care
Heparin Sodium	Northstar Rxllc.	72603-412-01	Intraveneous injection
Isoflurane	Baxter Anesthetic & Crit Care	1001936060	For anesthesia
Isopropyl alcohol swabs	BD	326895	Disinfect the skin before surgery
Microscope	Carl Zeiss	303294-9903	For mice microscopic surgery
Mini Trimmer	Braintree Scientfic	CLP-88	For mice hair removal
Optixcare Eye Lube Plus	AVENTIX	OPX-4252	Ophthalmic lubricant for eye protection during surgery
Pivetal Alloxate (Meloxicam)	PIVETAL	21294589	Analgesia
Portable Cauterizing Instrument Kit	McKesson Argent	42295143	For mice microscopic surgery
Ringer’s lactate buffered solution	Thermo Scientific	AAJ67572AP	For graft preservation
Small Animal Heated Pad	K&H	HM10	For mice surgery
Transpore White Surgical Tape	3M	7100115870	Fix the limb, head, and tail of mice during procedure
Vessal clamp	Fine Science Tools	00398-02	For mice microscopic surgery