A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

Summary

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

Abstract

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Introduction

A small animal model of heart transplantation (HTx) is critical to conducting research studying the pathophysiological conditions that affect the transplanted heart. Heterotopic HTx in a murine model, as described by Oto and Lindsey, has allowed researchers to study the pathophysiological changes observed in the conditions of ischemia and reperfusion¹. Traditionally, donor hearts for transplantation have been procured from beating heart donors, also known as donation after brain death (DBD) donors; however, there has been a disproportionate increase in the number of patients in need of HTx². More recently, hearts from ci....

Protocol

All animal experiments were conducted in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 86-23, revised 2011)⁸. The following procedures were approved by the Virginia Commonwealth University's Institutional Animal Care and Use Committee. All procedures were performed following OSHA (occupational safety and health administration) guidelines and the recommended sterile technique.......

Discussion

For a successful DCD heterotopic rat HTx, it is critical that a meticulous and thoughtful setup of the experiment is established. The detailed setup takes into consideration several factors, including 1) selecting young rats as DCD donors, 2) using isoflurane as the anesthetic agent of choice, 3) effective delivery of cardioplegia to the donor heart, 4) storage of the donor's heart in ice-cold solution, 5) limiting the abdominal dissection without compromising the lumbar vessels to only expose the infrarenal aorta an.......

Materials

Name	Company	Catalog Number	Comments
5-0 nylon suture polyamide monofilament	Aros Surgical	SP17A05N-45
5-0 silk suture	Surgical Specialties	SP116
8-0 monofilament suture	Aros Surgical	T06A08N14-13
Autoclave	Steris	Amsco Lab 250
BD Insulin Syringe with Detachable Needle 1 mL Syringes	Fisher Scientific	14-820-28
BD Syringe with Luer-Lok Tips (Without Needle) 10 mL Syringes	Fisher Scientific	14-823-16E
Belzer University of Wisconsin cold storage solution	Bridge to Life Northbrook IL USA		Adenosine 1.34 g/L, Allopurinol 0.136 g/L, Glutathione 0.922 g/L, Lactobionic Acid (as Lactone) 35.83 g/L, Magnesium Sulfate heptahydrate 1.23 g/L, Pentafraction 50 g/L, Potassium Hydroxide 5.61 g/L, Potassium Phosphate monobasic 3.4 g/L, Raffinose pentahydrate 17.83 g/L
Buprenorfin SR Lab	Zoopharm LLC
Debakey atraumatic pediatric multi-angle vascular clamp	Aesculap	F341T
Exel International Disposable Safelet I.V. Catheters 14 G	Fisher Scientific	14-841-10
Exel International Disposable Safelet I.V. Catheters 22 G	Fisher Scientific	14-841-20
Fogarty catheter size 4F	Edwands Lifesciences	120404F
Forceps with curved tips	Accurate Surgical & Scientific Instruments Corporation	ASSI.228
Gaymar Heating pump	Braintree Scientific, Braintree, MA, USA	TP700
Germinator-500	Braintree Scientific
Heparin Sodium Injection, USP 1,000 U/mL	Pfizer	NDC 0069-0137-01
Iris micro-scissors with straight tips	Accurate Surgical & Scientific Instruments Corporation	ASSI.5253
Isoflurane USP	Patterson Veterinary	NDC 14043070406
Ketamine HCl 100 mg/mL	Henry Schein	NDC 6745710810
Lidocaine HCl 2%	Aspen Veterinary	07-892-4325
McKesson General Medical 6IN Q-TIPS 2STER WOOD 100/PACK	Fisher Scientific	NC0650323	sterile cotton tip applicators
Micro-scissors, right angle and curved tips	Braintree Scientific	SC-MS 154
Needle holder	Accurate Surgical & Scientific Instruments Corporation	ASSI.BSL158	with the lock mechanism removed
Normal Saline	Baxter Infusion supplies
PowerLab station	AD Instruments, Denver, CO		data acquisition system
Sodium Hydroxide/Hydrochloric Acid			adjust the solution to pH 7.4
Sprague Dawley rats			male, 8–16 weeks of age, <400 g in weight
Surgical Microscope	Leika	Model M525 F40
Surgicel	Ethicon		absorbable hemostat
Temperature probe Therma Waterproof Type T High Precision Thermocouple Meter	Thermoworks	THS-232-107
Tweezers with high precision point	Excelta	17-456-109
Vecuronium Bromide	Sigma-Aldrich	PHR1627	diluted in PBS for 100 mg/mL
Ventelite	Harvard Apparatus, Holliston, MA, USA
Xylazine 100 mg/mL	Pivetal Anased	NDC 04606675002