Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Summary

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Abstract

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Introduction

Abbott et al. published¹ the first description of heterotopic abdominal heart transplantation in rats in 1964. These surgical techniques were refined and simplified by Ono et al. in 1969². Corry et al. first described a method for heterotopic abdominal heart transplantation in mice in 1973; similar to the previously reported rat models, this involved engraftment into the host's abdomen with revascularization by end-to-side anastomoses of the donor's pulmonary artery and ascending aorta to the recipient's inferior vena cava and abdominal aorta, respectively³. Heterotopic cervical heart ....

Protocol

All animal handling procedures were conducted in compliance with the NIH Care and Use of Laboratory Animals guidelines and approved by the Animal Studies Committee at Washington University School of Medicine. Hearts from C57BL/6 (B6) and BALB/c mice (weighing 20-25 g) were transplanted into gender-matched B6 recipients (6-8 weeks of age). The mice were obtained from commercial sources (see Table of Materials). Syngeneic transplants were performed to evaluate cellular responses related to ischemia-reperfu.......

Discussion

Utilizing this technique, mouse heterotopic cervical cardiac transplantation can be performed in less than 40 min by an experienced microsurgeon and in approximately 60 min by an entry-level microsurgeon. While cervical heart transplantation has been studied in numerous animal models, a mouse model remains the gold standard due to multiple well-defined genetic strains, genetic alteration capabilities, and the availability of numerous reagents, including monoclonal antibodies²⁴. The technique descr.......

Materials

Name	Company	Catalog Number	Comments
6-0 braided silk ties	Henry Schein Inc	7718729
0.75% Providone iosine scrub	Priority Care Inc	NDC 57319-327-0
10-0 nylon suture	Surgical Specialties Corporation	AK-0106
655-nm nontargeted Q-dots	Invitrogen	Q21021MP
70% Ethanol	Pharmco Products Inc	111000140
8-0 braided silk ties	Henry Schein Inc	1005597
Adson forceps	Fine Science Tools Inc	91127-12
BALB/c and C57BL/6 mice (6-8 weeks)	Jackson Laboratories
Bipolar coagulator	Valleylab Inc	SurgII-20, E6008/E6008B
Carprofen (Rimadyl) injection	Transpharm	35844
Carprofen (Rimadyl) oral chewable tablet	Transpharm	38995/37919
Custom-built 2P microscope running ImageWarp acquisition software	A&B Software
Dumont no. 5 forceps	Fine Science Tools Inc	11251-20
Fine vannas style spring scissors	Fine Science Tools Inc	15000-03
GraphPad Prism 5.0	Sun Microsystems Inc.
Halsey needle holder	Fine Science Tools Inc	91201-13
Halsted-Mosquito clamp curved tip	Fine Science Tools Inc	91309-12
Harvard Apparatus mouse ventilator model 687	Harvard Apparatus	MA1 55-0001
Heparin solution (100 U/mL)	Abraxis Pharmaceutical Products	504031
Imaris	Bitplane
Ketamine (50 mg/kg)	Wyeth	206205-01
Microscope—Leica Wild M651 × 6–40 magnification	Leica Microsystems
Moria extra fine spring scissors	Fine Science Tools Inc	15396-00
Ohio isoflurane vaporizer	Parkland Scientific	V3000i
Qdots	ThermoFisher	1604036
S&T SuperGrip Forceps angled tip	Fine Science Tools Inc	00649-11
S&T SuperGrip Forceps straight tip	Fine Science Tools Inc	00632-11
Sterile normal saline (0.9% (wt/vol) sodium chloride	Hospira Inc	NDC 0409-4888-20
Sterile Q-tips (tapered mini cotton tipped 3-inch applicators)	Puritan Medical Company LLC	823-WC
Surflow 20 gauge 1/4-inch Teflon angiocatheter	Terumo Medical Corporation	SR-OX2032CA
Surflow 24 gauge 3/4-inch Teflon angiocatheter	Terumo Medical Corporation	R-OX2419CA
ThermoCare Small Animal ICU System (recovery settings 3 L/min O2, 80 °C, 40% humidity)	Thermocare Inc
VetBond	Santa Cruz Biotechnology SC361931	NC0846393
Xylazine (10 mg/kg)	Lloyd Laboratories	139-236