Heterotopic Cervical Heart Transplantation in Mice

Summary

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Abstract

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient’s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

Introduction

The heart transplantation model is frequently used to investigate I/R-injury, allograft rejection, post-transplant vasculopathy and the efficiency of immunomodulating agents. Mouse models combine many advantages like the known immunological and genetic backgrounds, the availability of many inbred and transgenic strains and relatively low experimental costs.

In 1973, a technique of abdominal heart transplantation in mice was first described by Corry et al¹. In this model grafts are revascularized by end-to-side anastomoses of the ascending aorta to the recipient’s abdominal aorta and of the main pulmonary artery to the inferior vena cava. In 1991, a suture technique for cervical heart transplantation in mice was described by Chen et al². In this model a retrograde coronary artery perfusion is established by anastomosis of the recipient’s carotid artery to the ascending aorta of the graft. The venous blood drains via the coronary sinus into the right atrium, the right ventricle and is ejected through the pulmonary artery into the recipient external jugular vein (Figure 1). In comparison to the abdominal graft implantation the cervical model is less invasive and the superficial location of the graft enables a very easy accessibility for additional follow-up examinations (e.g. palpation, echocardiography, intravital fluorescence microscopy)^3,4.

Despite technical improvements^5,6, a more widespread use of this model has been limited due to technical difficulties to perform sutured anastomoses of small vessels with a high incidence of anastomosis leakage and thrombosis. In 1991, Matsuura et al. introduced the cuff technique in which the vessel wall is everted over a synthetic cylinder, which greatly facilitates the model of cervical heart transplantation⁷. The technique was further adopted for various experimental transplant models including renal⁸, pancreatic⁹, limb¹⁰, hepatic^11,12, lung^13,14 and vascular transplantation¹⁵. In comparison to the suture technique the required time for anastomoses and, thus, the warm ischemia time is shorter and there are significantly less vascular complications using the cuff technique¹⁶. Moreover, it enables surgeons with little training in microsurgery to perform the operation with a high success rate. A disadvantage of the cuff technique is the prerequisite ligation of the common carotid artery, which may alter cerebral perfusion.

In this video paper we present an improved and simplified cuff technique for cervical heart transplantation in mice.

Protocol

All experiments were approved by the German Heart Center Munich and the Government of Bavaria, Germany (Gz. 55.2-1-54-2532.3-17-13). Animals were housed in a specific pathogen free facility and received human care in compliance with the ”Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the ”Guide for the Care and Use of Laboratory Animals” provided by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985).
NOTE: A complete set of sterile microsurgical instruments, including two curved forceps and vessel dilatators, is necessary for the procedure (Figure 2a). Perioperative antibiotic prophylaxis is not routinely necessary but may be indicated in immunocompromised animals. Animals were sacrificed using terminal isoflurane inhalation.

Donor Preparation and Graft Harvest

1. Anesthetize the animal by intraperitoneal (i.p.) injection of midazolam (5.0 mg/kg), medetomidine (0.5 mg/kg) and fentanyl (0.05 mg/kg body weight). Place the mouse in a supine position on the operative field and confirm surgical tolerance by the pedal withdrawal reflex.
2. Disinfect the skin three times with chlorhexidine and perform a midline abdominal incision. Retract the intestines with wet gauze to the left to expose the inferior vena cava.
3. Inject 50 IU of heparin diluted in 0.4 ml saline through the inferior vena cava for anticoagulation. Incise the vessel 1 min after heparinization.
4. Open the diaphragm and perform a bilateral thoracotomy to expose the heart. The anterior chest wall is reflected superiorly and held in place with a clamp.
5. Remove the thymus and bluntly dissect the connective tissue between aorta and pulmonary trunk. Incise the right superior vena cava.
6. Puncture the mobilized ascending aorta at the level of the brachiocephalic trunk with a 30 G needle mounted on a 5 ml syringe and slowly perfuse the heart retrogradually without excessive pressure with 3 ml of 4 °C cold cardioplegic solution. Avoid the entry of air into the coronary circulation. Drop ice-cold saline solution onto the graft to further enhance cardiac arrest.
7. Transect the aorta proximally to the origin of the brachiocephalic trunk.
8. Ligate the inferior and both superior venae cavae close to the heart with 8-0 silk and transect them distally to the ligatures.
9. Dissect free the pulmonary trunk as distally as possible. Ligate the pulmonary arteries close to the bifurcation with 8-0 silk and transect them distally.
10. Ligate the pulmonary veins all together with a single 8-0 silk ligature and transect them.
11. Remove the heart from the donor and store it in ice-cold cardioplegic solution until implantation.

2. Recipient Preparation

1. Prepare the cuffs for anastomoses by cutting the polyimide tubing with a No. 11 scalpel or a scissor under the microscope. The cuff consists of a cylindrical body and an extension (¹/₃ of the circumference) for the placement of the vascular clamp, each of a length of 1 mm (Figure 2b).
2. Anesthetize the animal and confirm surgical tolerance as described above. Inject 0.25 ml warm saline solution subcutaneously (s.c.) for fluid replacement.
3. Remove the hair of the right cervical region using an electric shaver and place the animal in a supine position on a warm pad. Secure the legs with strands of tape while avoiding overstretching the front limbs as this can compromise respiration. Cover the eyes using ophthalmic ointment to prevent dryness. Disinfect the surgical site three times with chlorhexidine.
4. Make a transverse skin incision from the right mandibular angle to the jugular notch.
5. Bluntly mobilize the external jugular vein. Cauterize side branches with a bipolar forceps and transect them.
6. Divide the external jugular vein between ligatures at the level of the confluence with the vein from the submandibular gland.
7. Pull the external jugular vein through the cuff and occlude the vessel proximally by placing a vascular clamp on the extension of the cuff. Remove the ligature and irrigate the vessel lumen with 1:10 heparinized saline.
8. Evert the vessel wall over the cuff and fix it with a preset circumferential 8-0 silk ligature.
9. Remove the superficial part of the sternocleidomastoid muscle.
10. Mobilize the right common carotid artery and transect the vessel between ligatures below the carotid bifurcation.
11. Pull the carotid artery through the cuff and occlude the vessel proximally by placing a Yasargil clamp on the extension of the cuff. Remove the ligature and irrigate the vessel lumen with 1:10 heparinized saline.
12. Gently dilate the arterial lumen with a vessel dilatator, evert the vessel wall over the cuff and fix it with a preset circumferential 8-0 silk ligature.
13. To gain enough space for graft implantation, remove the right lobe of the submandibular gland.
14. Keep the operative field moist until implantation.

3. Graft Implantation

1. Place the graft in the recipient cervical region upside down with the aorta orientated medially and the pulmonary artery laterally.
2. Pull the aorta of the graft over the cuff with the carotid artery and secure the anastomosis with a circumferential 8-0 silk ligature.
3. Incise the pulmonary trunk on its anterior aspect and pull it over the cuff with the external jugular vein. Again secure the anastomosis with a circumferential 8-0 silk ligature.
4. Remove the clamp on the external jugular vein, following by unclamping of the carotid artery. The hearts fills immediately with blood. To enhance reperfusion drop 37 °C warm saline solution onto the graft. Normally the heart develops sinus rhythm within one minute. Remove the cuff extension at the venous site.
5. Ensure correct graft position, anastomoses must be tension-free and vessels without torsion. Do not cauterized vessels of the skin since this may impair wound healing. Bleeding usually stops spontaneously. Close the skin with a single 8-0 continuous suture.

4. Postoperative Care

1. Inject 0.25 ml warm saline solution s.c. for fluid replacement.
2. To antagonize narcotics inject naloxone (1.2 mg/kg), flumazenil (0.5 mg/kg) and atipamezol (2.5 mg/kg body weight) s.c. Left the animal on the warm pad until it has regained sufficient consciousness to maintain sternal recumbency. Observe the animal for at least one additional hour to register any abnormal behavior. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.
3. Provide preemptive analgesia with buprenorphine (0.05 mg/kg s.c. three times per day) or an alternative analgesia according to a local institutional protocol for a minimum duration of 72 hr after the procedure.
4. The surgeon and a veterinarian should see animals regularly. In case of weight loss >15%, apathy, swelling or surgical side infection animals are euthanized using terminal isoflurane inhalation.

Results

Our team could successfully perform the described technique after approximately 20 training surgeries. The most challenging part is to evert the vessel wall of the carotid artery over the cuff, because it easily flips back and multiple attempts may lead to tearing of the wall. After the training period, the mean total operative time was 60 ± 8 min. Around 20 min are required for donor harvest, 25 min for preparation of the cervical vessels and 15 minutes for anastomoses and skin closure.

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Discussion

We have completed over 200 cardiac transplants with a success rate over 90% in various experimental settings. With the technique described here, the complete procedure can be done within 60 min by an experienced surgeon. By preparing the cervical vessels before organ procurement, total graft ischemia time can be limited to 20 min. Time for implantation (warm ischemia time) is significantly shorter using the cuff technique compared to the suture technique¹⁶.

Instead of simply excisin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps JFC-7.2	S&T	P-00036	Curved tip 0.20 mm
Vessel dilatator D-5a	S&T	S-00124
Adventita scissor	S&T	S-00102
Vascular clamp B-1V	S&T	S-00396
Yasargil Clip	Peter Lazic	65,097	3.5 mm lenght
Bipolar forceps	Micromed	148-100-011	tip 0.25 mm
Polyimide tubes	River Tech Medical		19-24 gauge
8.0 silk ligatures	Catgut
Custodiol HTK solution	Essential Pharmaceuticals