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J Thorac Cardiovasc Surg 1994;108:1158-1159
© 1994 Mosby, Inc.

LETTERS TO THE EDITOR

A simplified blood-perfused isolated heart preparation

Quanmei Deng, MD, Alfonso G. Scicli, PhD


Carol Lawton

Norman A. Silverman, MD

Henry Ford Hospital
Division of Cardiac and Thoracic Surgery
2799 W. Grand Blvd.
Detroit, MI 48202

To the Editor:

Whether the experimental purpose is to evaluate long-term preservation, ischemia/reperfusion injury, coronary hemodynamics, or myocardial mechanics and metabolism, a crystalloid-perfused isolated small animal heart preparation has the advantages of low cost, precision of formulation, simplicity, and avoidance of thrombus formation However, clinical relevance will always be suspect because the role of erythrocytes, platelets, and leukocytes in oxidative stress and reperfusion injury are excluded, the lack of plasma proteins results in osmotic and oncotic inadequacies, and the baseline high oxygen tension and coronary flow rate are unphysiologic. For these reasons, we have developed a simple and reliable technique for perfusing isolated small animal hearts with autologous and homologous blood.

The experimental apparatus diagramed in Fig. 1 is set up before pairs of adult New Zealand rabbits (2.5 to 3.8 kg) are anesthetized and heparinized (2000 U). A 150 ml plastic cup with an oxygen catheter tubing serves as both the venous blood reservoir and the oxygenator. A siliconized glass perfusion column is used as a heat exchanger. Two transfusion filters (20 and 170 µm, Baxter 4c7700, Baxter Healthcare Corp., Irvine, Calif.), inserted before and after the roller infusion pump also facilitate debubbling, and a thermistor probe set into the perfusion line over the aorta monitors perfusate temperature. A second plastic cup is set in the glass organ chamber to collect blood drained from the left ventricular vent catheter. The perfusion system is initially primed with 100 ml of Ringer's solution, which is drained and discarded except for residual fluid in the tubing and filter. Blood collected from the blood donor rabbit is added to the venous reservoir and oxygenation is effected by bubbling the reservoir beaker with a mixture of 95% oxygen and 5% carbon dioxide to maintain oxygen tension between 120 and 200 torr and carbon dioxide tension between 35 and 45 torr. The pH is corrected to 7.35 to 7.45 with sodium bicarbonate.



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  		Fig. 1. Diagram of perfusion apparatus. 1, Siliconized glass column; 2, drug injection port; 3, perfusion pressure port; 4, pacing wires; 5, organ chamber; 6, pulmonary artery cannula; 7, intraventricular balloon; 8, left ventricular vent catheter; 9, blood reservoir; 10, oxygen catheter tubing; 11, 20 µm filter; 12, roller pump; 13, 170 µm filter; 14, water bath.

 
After tracheostomy, the animals' lungs are ventilated with room air. The abdomen of the blood-donor rabbit is opened and the abdominal aorta cannulated with an 18-gauge ball-end needle for collection of blood, which is added to the venous reservoir. The thorax and abdomen of the heart-donor rabbit are opened. Both the abdominal aorta and ascending aorta are cannulated in situ, and retrograde perfusion is initiated before the heart is harvested, as blood is withdrawn and added to the reservoir. The pulmonary artery is cannulated for collection and measurement of coronary effluent. The heart is then harvested and mounted on the column by switching the aortic perfusion line. During stabilization, the venae cavae are ligated, the lungs removed, and the heart instrumented with an intraventricular latex balloon for measurement of cardiac function. A 1 mm diameter plastic vent catheter is inserted, and the sinus node is crushed after placement of pacing wires. Because perfusion in situ has been established, hearts do not stop beating and ischemia is minimized during harvesting and suspension. A sidearm syringe infusion pump allows administration of pharmacologic agents to measure endothelium-dependent and -independent coronary flow reserve.

Suspension of the heart is successful if the developed pressure is 80 mm Hg or greater after 40 minutes of equilibration. In the last 112 preparations, only three were hemodynamic failures. Hemodynamic performance and coronary blood flow were constant for 3 hours (Fig. 2), and there was no evidence of thrombus formation in the circuitry despite no additional heparin administration. (Data are given as mean ± standard error of the mean.) Systolic performance (developed pressure = 118 ± 11 mm Hg, maximum rate of pressure rise = 1825 ± 204 mm Hg/sec) was comparable to our previous experience with Krebs-Henseleit perfusates. The unstimulated coronary flow at this workload of 4.4 ± 0.4 ml/min per gram dry weight was fourfold to sevenfold less than with crystalloid perfusion. However, the percentage increase in coronary flow to endothelium-dependent (125 ± 14%) and -independent (84% ± 11%) agonists was physiologically as well as statistically significant. The perfusate composition of these experiments was as follows: hematocrit 25% to 30%, potassium 3.5 to 5.5 mEq/L, sodium 130 to 140 mEq/L, and ionized calcium 12 to 16 mg/dl (0.8 ml of 10% calcium chloride is empirically added to the venous reservoir on initiation of perfusion).



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  		Fig. 2. Representative systolic performance of a blood-perfused heart showing constant developed pressure for 3 hours of perfusion.

 
We believe this modified perfusion technique has distinct advantages over previously reported preparations. Unlike the method of Lorell and associates,Go 1 purchase of a membrane oxygenator is not necessary and, if the blood is bubbled with a 0.5 mm diameter tubing, relatively large bubbles minimize foam development and hemolysis yet provide adequate oxygenation. Moreover, adhesive reaction of the neutrophils to the glass wall is avoided by siliconizing the perfusion column. Perfusing the heart by cross-circulation with a support animal is both time-consuming and technically demanding.Go 2 Perhaps more important, it is impossible to isolate the heart from the influences of pharmacologic agents administered to maintain the support animal, varying levels of anesthesia, and the neuroendocrine response to trauma. Yaku and colleaguesGo 3 have described rabbit heart perfusion with a suspension of bovine red cells in crystalloid buffer. However, this requires availability of freshly slaughtered animals and the unknown effects of heterologous perfusion. Furthermore, as constituted, the perfusate contains no leukocytes, whose important role in ischemia/reperfusion injury is increasingly evident. Given the distinct physiologic superiority of obtaining data from blood-perfused isolated heart preparations, it behooves investigators to develop simple, reproducible, and reliable experimental preparations. We believe the described technique fits that description.

References

  1. Lorell BH, Isoyama S, Grice WN, Weinberg EO, Apstein CS. Effects of ouabain and isoproterenol on left ventricular diastolic function during low-flow ischemia in isolated, blood-perfused rabbit hearts. Circ Res 1988;63:457-67.[Abstract/Free Full Text]
  2. Qiu Y, Hearse DJ. Comparison of ischemic vulnerability and responsiveness to cardioplegic protection in crystalloid-perfused versus blood-perfused hearts. J THORAC CARDIOVASC SURG 1992;103:960-81.[Abstract]
  3. Yaku H, Slinker BK, Myhre ESP, Watkins MW, LeWinter MM. Stability of myocardial O2 consumption–pressure-volume area relation in red cell–perfused rabbit heart. Am J Physiol 1991;30:1630-5.



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