The rabbit autoperfusing heart-lung preparation

JOURNAL

OF SURGICAL

RESEARCH

4,

104-108 (1988)

The Rabbit Autopetfusing ALAN MUSKETT,

Heart-Lung

Preparation

M.D., NELSON A. BURTON, M.D., MICHAEL GROSSMAN,M.D., AND WILLIAM A. GAY, JR., M.D.

Division of Cardiothoracic Surgery, Department of Surgery, University of Utah Medical Center, 50 North Medical Drive, Salt Lake City, Utah 84132 Submitted for publication November 5, 1986 The autoperfusing heart-lung preparation holds promise as a method for extended organ preservation. Further understanding of the hemodynamics, metabolism, and quality of tissue preservation of this preparation may increase the number of available organs for heart-lung transplantation. We describe the materials and operative technique for a small animal model of the autoperfusing heartlung preparation. The method is easy to learn, the materials and animal subjects are inexpensive, and the preparation is fully monitored to provide consistent and reproducible data. The rabbit autoperfusing heart-lung preparation is an excellent vehicle for the investigator studying warm organ preservation. 0 1988 Academic Fxss, Inc.

A platelet transfer pack (Fenwal, code 4R2014) servesas a good reservoir. It comes The autoperfusing heart-lung preparation with an attached length of intravenous-type has been used successfully as a method of tubing which, when trimmed to 20 in., serves organ block preservation in human heartas an arterial line. A second line with atlung transplantation [ 11. Although cumbertached coupler (Fenwal Plasma Transfer Set, some for short-term preservation, the prepacode 4C2240) is inserted into the reservoir. ration holds promise for extended extracorThe middle port of the reservoir is cannuporeal organ function if significant issues of lated with an injector port (Fenwal Sampling hemodynamics, metabolism, and quality of Site Coupler, code 4C2405) for sampling of preservation can be systematically addressed. reservoir blood. Small tubing adapters are We present an easily reproducible, inexpeninserted in the cut ends of the arterial and sive small animal model for the autoperfusvenous lines and plugged into three-way ing heart-lung preparation. stopcocks. Pressure tubing is connected to the lateral port of the stopcocks for monitorTHE PERFUSION APPARATUS (FIG. 1) ing of arterial and venous pressures. The The function of the perfusion apparatus is cannulae (DLP, Inc., Vessel Cannula No. to approximate the normal cardiopulmonary 3000 1) are those used for inflating saphenous environment as closely as possible. The heart veins being prepared for coronary artery surejects blood through an aortic cannula which gery. The tapered ends of the cannulae are is attached by tubing to an elevated reservoir. trimmed to provide a larger lumen. They are The height of the reservoir determines the connected directly to the stopcocks. A C aortic perfusion pressure, or afterload. clamp is placed on the venous tubing for regThrough a second line attached to the reser- ulation of right atria1 return. The reservoir is primed with a small voir, the right atrium receives “venous” return regulated to maintain a normal right amount of heparinized saline and the lines atrial pressure (preload). Pulmonary blood are flushed and clamped. The reservoir is flow continues, and the perfusate is oxygen- suspended from a clamp attached to a ringstand. A heating lamp is connected to the ated and ventilated by the lungs. INTRODUCTION

0022-4804/88 $1SO Copyright 0 1988 by Academic Press, Inc. AU rights of reproduction in any form resrvd.

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MUSKETT ET AL.: AUTOPERFUSING

HEART-LUNG

105

PREPARATION

PRESSURE-BAG ‘WITH I” SOLUTION

PLASTIC SAPHENOUS VEIN CARD,0 CANNUL p.i,. PRESSURE LINE ISFRUACI

MULTI-PoRT,YPNIFOLD, LINE HOOK-UP

HlG” PRESSURE

u

1

FIG. 1. The perfusion apparatus.

ringstand above the reservoir and used as needed to help regulate temperature. Pressurelines for left atrial and pulmonary artery pressures are also prepared. The left atria1 cannula is the same used for aortic and right atria1 pressures, while a flexible 3 French umbilical catheter is used for the pulmonary artery. All pressure lines are to a stopcock console interfaced with a transducer, which is then connected to a digital monitor. A 2-liter beaker is filled with water warmed to 37°C with an aquarium heater. A 250~ccbeaker is suspended inside the larger container and connected to the ringstand with a large clamp. The smaller beaker is partially filled with saline and serves as a bath for the heart-lung block. A thermometer is used to monitor the bath temperature. The suspension system is shown in Fig.. 2.

with careful titration of sodium pentobarbital (30 mg/kg) through a marginal ear vein until the animal no longer responds to painful stimuli. A transverse incision is made two finger breadths above the sternal notch. The trachea is isolated and a 4-mm metal cannula is inserted. A Y connecter is attached to the cannula which is in turn connected by plastic tubing to a rodent ventilator (Harvard, Model 683). Ventilation is begun at 10 AHLP

SUSPENSION

SYSTEM

SUSPENSIOW GAR WLTH ,sPrtl”G LOADED CLAMP FORHEART-LUNGBLOCK

EATING ELEYENT

EXPLANATION

OF THE PROCEDURE

Larger rabbits (3.0-4.5 kg) are preferred due to larger vessel size and are used after approval by the institution’s Animal Care Committee. Ketamine (50 mg/kg) is administered intramuscularly prior to preparation. The upper abdomen, thorax, and neck are shaved. Deeper anesthesia is then obtained

FIG.

2. The suspension system.

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1988

cc/kg at a rate of 30/min. Room air can be used as a gas mixture, or room air combined with a 10% COz to maintain a more physiologic PC02 once the block is removed. The cannula is secured to the distal trachea with a heavy tie and the trachea is transected. A median sternotomy is then performed. Heavy scissors divide the sternum nicely. Care must be taken to avoid the left innominate vein. Gelpi or Wheatlander retractors provide good exposure. The thymus is dissected free from the underlying mediastinal structures using a Q-tip (Fig. 3). The base of the thymus is divided and the organ is removed. The aortic arch and branch vessels are identified and isolated. Proximal and distal 4-O silk ties are placed loosely on the right subclavian, right FIG. 4. (A) Isolation of the great vesselsprior to cancarotid, and left carotid arteries. A heavy tie nulation. (B) Partial occlusion of the aortic arch prior to encircles the aortic arch just proximal to the cannulation of the aorta through the base of the innomileft subclavian artery. A 2-O silk is placed in nate artery. position at the base of the innominate artery. The right and left superior cavae and the inferior vena cava are then loosely encircled placed in the right and left atria and at the with 4-O silk ties proximally and distally. base of the pulmonary artery (Fig. 4A). Using 5-O monofilament sutures on small The animal is then heparinized with 1000 tapered needles, purse string sutures are units of sodium heparin per kilogram injected into the right atria1 appendage using a 27-gauge needle. The left subclavian, left carotid, and right carotid arteries are ligated and divided. A bulldog clamp is placed across the top of the aortic arch just below the innominate artery (Fig. 4B). A small transverse incision is made in the innominate using an 11 blade and the arterial cannula is inserted. The bulldog clamp is carefully releasedand the cannula tip is guided to about 1 cm above the aortic valve. The cannula is then tied securely at the base of the innominate and the vessel is divided. After cannulation, the arterial line is then unclamped, allowing ejection into the reservoir. \ The distal aorta is then ligated, completely diverting output into the reservoir. The right atria1 cannula is then placed through a small incision in the appendage and secured with the purse string suture. At this point, the reFIG. 3. Dissection of the thymus from the heart and spiratory rate is slowed to four breaths per great vessels.

MUSKETT ET AL.: AUTOPERFUSING

HEART-LUNG

UTILITY

PREPARATION

107

OF THE PREPARATION

Our experience with this preparation now includes over 100 experiments. After overcoming the technical challenges of this small animal model, it is reliably reproducible. Once the preparation is underway, it requires very little maintenance. Bath temperature, blood gases,and blood glucose should be monitored periodically. We use a 50% glucose solution as needed to maintain blood sugar greater than 100 mg/dl. No hemodynamic adjustments should be required after initial stabilization. In order to assessthe quality and duration of organ survival, we have arbitrarily defined FIG. 5. The heart and lungs are retracted superiorly as a functional preparation as one able to the organ block is dissected free of the posterior mediasmaintain a systolic blood pressure of 60 mm tinum. Hg after initial stabilization at a right atria1 pressure of 5- 10 mm Hg and a systolic pressure of greater than 60 mm Hg. In addition, minute. After the reservoir volume stabilizes, the pOZ must be greater than 80 Torr and the the cavae are ligated and divided. The C 60, less than 40 Torr on room air ventilaclamp is adjusted to maintain a right atria1 tion. Usually the pCOZ is much lower bepressure of 5- 10 mm Hg. The left atria1 and cause the only CO2 produced is that by the pulmonary artery pressure lines are then heart and lungs. placed and secured. The reservoir height is adjusted to maintain a systolic aortic pressure of 55-65 mm Hg. The inferior pulmonary ligaments are divided. Dissection between the esophagusand the posterior lung is meticulous to avoid bleeding or air leaks. The surgeon can use the middle and index fingers to retract the organs caphalad while dissecting with the other hand (Fig. 5). Care must be taken to divide the descending aorta distal to the tie. After the organ block is freed, it is placed in a plastic bag (Fig. 6). The bag is loosely tied around the perfusion and monitoring lines. The organs are placed in the water bath with a clamp around the metal tracheal cannula supporting the preparation vertically. The pressure lines are then recalibrated and the batch and reservoir temperatures are checked. The reservoir height is readjusted. Initial blood gasesand other studies can then be performed by sampling blood from the FIG. 6. After the organ block is removed, it is placed in reservoir injection port. a plastic bag and then in the water bath.

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Using these criteria, the preparations survive a mean of 4.36 hr when left alone. We have found that treatment of the preparation with intravenous indomethacin prior to incision (0.2 mg/kg) increases the survival to a mean of 7.36 hours (range 5.5-l 1.0 hours) [2]. The untreated preparations failed due to severepulmonary hypertension with secondary right ventricular failure, which developed at about four hours. Treatment with indomethacin resulted in significantly lower pulmonary artery pressures,possibly by inhibiting the formation of thromboxane, a potent mediator of vasoconstriction and platelet aggregation. Activation of platelets and leukocytes, with their subsequent release of thromboxane, may occur as a result of contact with the foreign surfacesof the perfusion apparatus. Currently, we are testing the efficacy of platelet and leukocyte depletion (of the perfusate blood) on the quality and duration of preparation survival. Some preparations have survived beyond 12 hr with good organ function. While these preparations do not survive as long as the bovine preparation [3], they are fully functional and respond to intervention. Becausethe organs are small and the preparation is delicate, changes may occur more acutely. However, the changesappear to represent the natural history of an autoperfusing system, making this a useful model. Although distance procurement of heartlung blocks is currently accomplished by a cold crystalloid flush technique, this preparation may be useful in the future for periods of

1988

transport exceeding 4 hr. As the search for organ donors broadens, warm nonischemic organ preservation for extended periods may have a significant role. More immediate roles for the preparation may be as a model in the study of cardiopulmonary interaction in the absence of exogenous neuroendocrine influence or as a model of pulmonary hypertension. CONCLUSION

The rabbit model for the autoperfusing heart-lung preparation has several positive features. Repetitive experiments involving the hemodynamics, metabolism, and survival of the preparation can be performed at a minimal cost. One or two persons can perform the procedure, which is easily learned. It is a consistent, reliable preparation which may aid in the study of heart-lung interaction, pulmonary hypertension, and organ preservation. REFERENCES Ladowski, J. P., Kapelanski, D. P., Teodori, M. F., Stevenson, W. C., Hardestry, R. L., and Griffith, B. P. Use of autoperfusion for distance procurement of heart-lung allografis. Heart Trunspkzn?.4: 330, 1985. Musket& A. D., Burton, N. A., Gay, W. A., Miller, M., and Rabkin, M. S. Preservation in the rabbit autoperfusing heart-lung preparation: A potential role for indomethacin. Surg. Forum 37: 252, 1986. Robicsek, F., Masters, T. N., Duncan, G. D., Denyer, M. H., Rise, H. E., and Etchison, M. An autoperfused heart-lung preparation: Metabolism and function. Heart Transplant. 4: 334, 1985.