New Experimental Technique to Study Blood Cardioplegia in the Isolated, Perfused Rat Heart Mukul Chandra, MD, Herzl Schwalb, PhD, Ester Yaroslavsky-Houminer, BA, Yori Appelbaum, MSc, Gideon Uretzky, MD, and Joseph B. Borman, MD The Joseph Lunenfeld Cardiac Surgery Research Center, Hadassah University Hospital, Jerusalem, Israel

Blood cardioplegia has been extensively studied clini- cally and in the large animal experimental model. We describe here a modification of the original Langendorff technique to study continuous warm blood cardioplegia in the isolated, perfused rat heart. The excised heart is mounted on the perfusion apparatus and perfused with Krebs-Henseleit buffer. Prearrest hemodynamics are re- corded. The shed blood in the mediastinal cavity (8 to 12 mL) is collected, filtered, and reconstituted into car- dioplegic solution (hematocrit, 0.20; K+, 15 mmoYL). Hearts are arrested and maintained at 37OC by continuous recirculation of blood cardioplegia for 1 hour. The blood cardioplegia system consists of a Silastic tubing oxygen- ator, peristaltic pump, and two filters (40 pm pore size).

eports have appeared in the literature indicating the R superiority of warm blood cardioplegia over existing methods of myocardial protection [1-4]. Some of these studies have relied on the assessment of the human heart at the time of open heart operation. Control of variables in this situation is difficult because cardiac pathology in itself may alter hemodynamic and metabolic responses after bypass. Experimental studies on intact large animals have been performed [5-71. They are infinitely easier to control than the human; nevertheless, there are many parameters that are difficult to keep constant and that require a large number of experiments with the concomitant consider- able expenditure for dogs and extracorporeal apparatus. The original method of Langendorff [8, 91 has been extensively applied to the study of crystalloid cardioplegic solutions. It has been the workhorse of most experimental myocardial metabolism studies to date [lo].

The Langendorff system offers the advantages of sim- plicity, stability, and the feasibility of studying a large number of hearts under controlled hemodynamic condi- tions. Although studies on isolated, perfused small ani- mal hearts with a support animal have been described [ll, 121, they did not use continuous warm blood cardioplegia as a method of myocardial protection.

The major benefits of blood cardioplegia are related to its ability to transfer oxygen and metabolites to the ar- rested myocardium, its buffer capacity, and its appropri-

The heart is reperfused with Krebs-Henseleit solution, and postarrest hemodynamics are recorded. Percentage recovery of peak left ventricular pressure, heart rate, and coronary flow were 98.5 2 3.1, 202 f 4.2, and 98.5 +: 4.5 (mean -t standard error of the mean; n = 61, respectively. Myocardial oxygen consumption during arrest was 57 pL - min-' - g-' dry wt, which is 10% of the myocardial oxygen consumption of a beating heart in in-vivo and ex-vivo models. These results suggest the feasibility of studying blood cardioplegia in the isolated, perfused rat heart model under controlled conditions. Continuous warm blood cardioplegic arrest provided excellent myo- cardial protection for 1 hour in this model.

(Ann Thorac Surg 2993;55:946-9)

ate osmotic environment for the myocardial cells. All of these major characteristics attributable to blood are opti- mal at normothermia.

We describe here an experimental model to study blood cardioplegia in an isolated, perfused rat heart apparatus in a manner that closely resembles the methodology in the operating room. This model has been used to compute the myocardial oxygen consumption during continuous warm blood cardioplegic arrest. The experimental tech- nique relies essentially on the concept of using the shed blood in the mediastinal cavity of the rat after the heart has been excised, reconstituting it into blood cardioplegic solution and perfusing it continually to arrest the heart. The cardioplegic solution is oxygenated in a Silastic tubing oxygenator and propelled by a peristaltic pump. Perfu- sion before and after cardioplegia is maintained by retro- grade perfusion of the heart on the Langendorff column with modified Krebs-Henseleit (KH) solution at a pressure of 90 cm H,O and a temperature of 37°C. This model can serve as a prototype for a variety of myocardial metabo- lism experiments in which blood is used as the vehicle for cardioplegia.

Material and Methods Animal Cure All animals have received humane care in compliance with the "Guide for the Care and Use of Laboratory

(NIH publication no. 85-23, revised 1985).

Accepted for publication July 24, 1992.

Address reprint requests to Dr Schwalb, Cardiac Surgery Research Center, Hadassah University Hospital, P O B 12000, Jerusalem 91120, Israel.

published by the Institutes Of

0 1993 by The Society of Thoraac Surgeons 0003-4975/93/$6.00

Ann Thorac Surg 1993;55:946-9

CHANDRA ET AL 947 BLOOD CARDIOPLEGIA IN ISOLATED HEART

Experimental Design Hearts were obtained from Sprague-Dawley rats weigh- ing 350 to 400 g. Half an hour before the removal of the hearts all rats received sodium heparin (1,500 IU, intraperitoneally). Under pentobarbital anesthesia (30 mgkg) the heart was excised and immediately placed in iced saline solution. After cessation of contraction the aorta was cannulated and the heart mounted on the Langendorff perfusion apparatus. Retrograde aortic per- fusion was maintained with KH solution containing the following (in mmoVL): NaCl, 118; KCl, 4.9; CaCl,, 3.0; MgSO,, 1.2; KH,PO,, 1.2; sodium EDTA, 0.5; NaHCO,, 25.0; glucose, 11.1; and heparin, 500 U/L. The perfusate was aerated with a mixture of 95% 0, and 5% CO,.

After the removal of the heart, the shed blood in the mediastinal cavity was aspirated in a heparin-flushed 20-mL syringe. Care was taken to avoid stray hair and other pieces of tissue. The volume of blood recovered was 8 to 12 mL. An equal amount of blood is drawn from another rat. Cardioplegia was reconstituted by mixing blood and St. Thomas' solution in a ratio of 1:l. The operating volume for the blood cardioplegia circuit is 35 to 40 mL, which constitutes the prime. The hematocrit can be vaned at discretion simply by altering the ratio of blood and crystalloid. A 1:l dilution results in a hematocrit of approximately 0.20, which is similar to our clinical prac- tice. Potassium chloride (15%) was added to bring the [K+] to 15 mmoVL. The overall composition of the blood cardioplegic solution was as follows: K+, 15 mmoYL; Na+, 135 mmol/L; C1-, 135 mmoVL; Ca2+, 1.8 mmol/L; and glucose, 4.4 mmoYL. Modifications were made in the Langendorff perfusion system to accommodate a peristal- tic pump, a Silastic tubing oxygenator, heat exchanger system, and filters for closed circle blood cardioplegia delivery at the desired temperature and flow.

Perfusion With Krebs-Henseleit Solution Oxygenated KH flows from the thermostated perfusion column to the heating chamber H, and through open tap T to the aortic cannula; it exits from the right heart into a beaker, from which it is disposed of (Fig 1). While the heart is being perfused by the crystalloid solution, the blood cardioplegia is circulated for the initial oxygenation and to trap any small thrombi before they encounter the coronary circulation. During this period the outlet from the blood cardioplegia circuit is disconnected from tap T and put into the thermostatically controlled heart cham- ber. This enables recirculation and aeration of the car- dioplegic solution. The oxygenator is composed of 120- cm-long Silastic tubing (0.058 inch inner diameter; Dow Corning, Midland, MI) suspended in a glass bottle with an entry and exit port for 0, and CO,. The blood cardioplegic solution is pushed by a small roller pump (Minipuls 2; Gilson, France) to the heating chamber H, through filters F,, F,, and F, (F, and F, are 40-pm pore size filters, whereas F, is a blood set filter).

Blood Cardioplegia Perfusion Circuit The reconstituted blood cardioplegia is placed in the heat-jacketed heart chamber. It flows through the Silastic

021 c02

Langendorff Reservoir

9 0 cm

Hz Hi

T

Pump

Oxygenator

1

Hernodynamic Recorder

W Fig 1 . lsolated rat heart preparation for continuous blood cardioplegia recirculation. For details, see Material and Methods section of text. (F = filter; H = heat exchanger; T = tap.)

tubing oxygenator and is propelled through the peristaltic pump into the blood reservoir mounted at a height of 90 cm.

Switching the tap T in the direction of blood cardiople- gia initiates cardioplegic perfusion and at the same time halts perfusion with KH. Blood cardioplegia flows through filters F, to F, and heat exchanger H,. It enters the aortic root and exits from the right side into the heart chamber and is recirculated. Cardioplegic arrest is initi- ated with a coronary flow rate of 4 to 5 mUmin and maintained at 3 mumin. The heart is immersed in the blood cardioplegia during the period of arrest to prevent the temperature from drifting. All glass surfaces in contact with blood were siliconized.

Intraventricular pressure is measured by a pressure transducer connected to a small latex balloon-tipped cath- eter inserted into the left ventricle through the left atrium. It is inflated to give an end-diastolic pressure of 0 mm Hg. These parameters are recorded on a multichannel re- corder (Hewlett Packard 7758B).

Hearts were subjected to 20 minutes of KH perfusion for stabilization. Hemodynamics and coronary flow were recorded. This was followed by 1 hour of cardioplegic arrest with continuous nomothermic blood cardioplegia

948 CHANDRA ET AL BLOOD CARDIOPLEGIA IN ISOLATED HEART

Ann Thorac Surg 1993;55:94&9

at a rate of 3 mL/min. The heart was reperfused with KH for 15 minutes, and hemodynamic recovery was evalu- ated. Blood gas analysis was done from samples at the aortic root and coronary sinus ends at the end of 30 minutes of cardioplegic arrest. Myocardial oxygen con- sumption (MvO,) was calculated as described [13]:

MvOz = [(CaOz - CvOz) x CF x lO]/dry wt

Ca02 = Hb x 1.36 x SVOZ + PaO2 x 0.003

C V O ~ = Hb X 1.36 X SVOZ + PVOZ X 0.03

where CaO, and CvO, = arterial and venous oxygen content, CF = coronary flow, Hb = hemoglobin concen- tration, SvO, = venous oxygen saturation, and PaO, and PvO, = arterial and venous oxygen tension. Plasma hemoglobin concentration of the blood cardioplegia was measured by the cyanmethemoglobin method [ 141 after circulation for 1 hour.

Results After 1 hour of cardiac arrest with continuous warm blood cardioplegia and 15 minutes of reperfusion with KH, the hemodynamic recovery was as follows: peak left ventric- ular systolic pressure, 105 f 4.6 mm Hg (98.5%); heart rate, 253 f 14.6 beats/min (102%); and coronary flow, 13.5 f 0.5 mL/min (97.5%) (figures in parenthesis are percent- ages of prearrest values; mean f standard error of the mean; n = 6). No change in left ventricular end-diastolic pressure was recorded throughout the experiment.

Blood gas analysis and oxygen consumption during cardiac arrest were as follows: arterial and venous oxygen tension were 206.6 f 21.6 and 121.5 f 15.6 mm Hg, respectively. Arterial and venous oxygen saturation were 99.6% f 0.12% and 97.4% -C 0.3%, respectively. The arterial oxygen content of blood was 8.7 t 0.06 mL/dL. After myocardial extraction it fell to 8.22 f 0.04 mL/dL on the venous side. Plasma hemoglobin level was 124.5 mg/dL (range, 85.4 to 178.5 mg/dL). Arterial and venous pH were 7.33 t 0.19 and 7.22 f 0.18, respectively. Myocardial oxygen consumption was 57.2 f 7 pL - min-' *g-'. Coronary flow was 3.0 f 0.0 mWmin, and dry weight was 0.26 f 0.02 g.

Comment The aim of this study is to introduce a conceptually novel experimental technique for studying blood cardioplegia in the isolated perfused animal heart system. An additional circuit using an oxygenator and a peristaltic pump has been incorporated to arrest the heart with blood cardio- plegia and maintain it by continuous perfusion.

After 1 hour of cardiac arrest with continuous warm blood cardioplegia, the left ventricular pressure and heart rate showed a complete recovery to prearrest levels. Electromechanical activity resumed almost immediately upon reperfusion. Reperfusion time to attain full hemo- dynamic recovery was less than 10 minutes. This can be explained by the fact that the heart was preserved at normothermia. No diastolic dysfunction was observed as

left ventricular end-diastolic pressure did not change from its prearrest value of 0 mm Hg.

The calculated value of MvO, in the normothermic arrested heart in our system is 57 pL - min-' - g-' dry weight. Buckburg and associates [15] found that myocar- dial oxygen consumption is 5.6 mL - 100 g-' - min-' in the beating, empty heart at 37°C but only 1.1 mL * 100 g-' - min-' in the arrested left ventricle, a decrease of more than 80%. Others [16] have worked out figures of 90% reduction in oxygen demands of the normothermic, arrested heart. In a study comparing myocardial protec- tion with intermittent cold blood cardioplegia versus continuous warm blood cardioplegia [17], MvO, was considerably lower in the continuous warm blood cardio- plegia group during the arrest period, despite the differ- ence in temperature (37" versus 15°C). This suggests an oxygen debt build up between boluses in the intermittent cold blood cardioplegia group. These results demonstrate that the major determinant of MvO, is electromechanical arrest, and once arrest is achieved the MvO, decreases to low levels. In a recent study Murashita and colleagues [lo] computed values of oxygen consumption in the isolated, working rabbit heart using asanguineous crystalloid solu- tions. The values range between 500 to 900 pL - min-' 'g-' dry weight in an in vitro model and 500 to 700 pL * min-' - g-' dry weight in vivo at physiological work loads. The level of oxygen consumption in the arrested heart of our model comes to about lo%, a figure in agreement with existing data [15, 161.

The described model provides accurate information of events occurring during the period of cardiac arrest (eg, MvO,, lactate production, pH). Experimental groups in which a single intervention is introduced during arrest (supplements to blood cardioplegia, change in hemat- ocrit, flow rate, temperature), may be compared with each other. In these situations the functional and metabolic status of the groups during reperfusion can be analyzed and statistically compared. These, however, cannot be accurately compared with those occurring either before or after arrest due to the differing nature (blood versus crystalloid) of the perfusate.

In a recent study [ll] of blood-perfused rabbit heart with a support animal, the ischemic vulnerability and cardioplegic protection were compared with a crystalloid perfused group. It was found that the former group had a greater resistance to ischemia and a superior response to cardioplegic solutions. The authors of that study con- cluded that the important factors contributing to the overall performance of the blood perfused preparation were oncotic and osmotic profile of blood and the pres- ence of erythrocytes.

It should be emphasized at this point that fastidious attention to the cleanliness of the system is essential to achieve consistent results. Before beginning the protocol and after finishing it we continually flush the cardioplegia delivery system for 30 minutes with distilled water with- out recirculation to get rid of microscopic particles. Blood cardioplegia is also recirculated for 15 minutes in a closed system before it perfuses the heart.

The optimal conditions for delivery of blood cardiople-

Ann Thorac Surg 1993:5594&9

CHANDRA ET AL 949 BLOOD CARDIOPLEGIA IN ISOLATED HEART

gia have been recently described “1. Maximal myocar- dial recovery was at a hemoglobin concentration of 80 glL. In our model we got excellent hemodynamic recovery of hemoglobin at 60 g/L, corresponding to a hematocrit of 0.20. Technically, the blood-perfused models may be more cumbersome and expensive. These disadvantages are, however, offset by the reliability of data obtained. Experiments may be performed with greater relevance to clinical studies.

This work was supported by grants from the Aaron Beare Foundation, South Africa, and the Samuel Lunenfeld Charitable Foundation, Canada.

References 1. Lichtenstein SV, Ashe KA, Dalati ED, Cusimano RJ, Panos

A, Slutsky AS. Warm heart surgery. J Thorac Cardiovasc Surg 1991;101:269-74.

2. Engelman RM. Retrograde continuous warm blood cardio- plegia. Ann Thorac Surg 1991;51:18&1.

3. Salerno TA, Houck JP, Barrozo CAM, et al. Retrograde continuous warm blood cardioplegia: a new concept in myocardial protection. Ann Thorac Surg 1991;51:2457.

4. Rozenkranz ER, Buckburg GD, Laks H, Mulder DG. Warm induction of cardioplegia with glutamate enriched blood in coronary patients with cardiogenic shock who are dependent on inotropic drugs and intraaortic balloon support: initial experience and operative strategy. J Thorac Cardiovasc Surg

5. Follette DM, Steed DL, Foglia R, Fey K, Buckburg GD. Advantages of intermittent blood cardioplegia over intermit- tent ischemia during hypothermic aortic clamping. Circula- tion 1978;58(Suppl 1):200-9.

6. Buckburg GD, Hottenrott C. Ventricular fibrillation. Ann Thorac Surg 1975;20:7&85.

1983;86:507-18.

7. Rozenkranz ER, Vinten-Johanssen L, Buckburg GD, Oka- mot0 F, Edwards H, Bugyi H. Benefits of normothermic induction of cardioplegia in energy depleted hearts, with maintenance of arrest by multidose cardioplegia infusions. J Thorac Cardiovasc Surg 1982;84:667-76.

8. Langendorff 0. Untersuchungen am uberlebenden Saugeth- ier herzen. Pflugers Arch 1895;61:291-332.

9. Neely JR, Rovetto MJ. Techniques for perfusing isolated rat hearts. Meth Enzymol 1975;1975:4?-60.

10. Murashita T, Kempsford RD, Hearse DJ. Oxygen supply and oxygen demand in the isolated working rabbit heart perfused with asanguinous crystalloid solution. Cardiovasc Res 1991; 25:198-206.

11. 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;103:96&8.

12. Gamble WJ, Conn PA, Kumar AR, Plenge R, Munroe RG. Myocardial oxygen consumption of blood perfused, isolated rat heart. Am J Physiol 1970;219:604-12.

13. Nightingale P. Practical points in the application of oxygen transport principles. Intensive Care Med 1990;16(Suppl 2): 973-7.

14. Brown BA. Hematology: principles and procedures; fifth ed. Philadelphia: Lea & Febinger, 198879-88.

15. Buckburg GD, Brazier JR, Nelson RL, Goldstein SM, Mc- Connell DH, Cooper N. Studies on the effects of hypother- mia on regional myocardial flow and metabolism during cardiopulmonary bypass. I. The adequately perfused beat- ing, fibrillating, and arrested heart. J Thorac Cardiovasc Surg 1977;73:87-94.

16. Bernard WF, Schwartz HF, Malick NP. Selective hypother- mic arrest in normothermic animals. Ann Surg 1961;153: 43-51.

17. Panos A, Kingsley SJ, Salerno TA, Lichtenstein SV. Contin- uous warm blood cardioplegia. Surg Forum 1990:23>5.

18. Yau TM, Weisel RD, Mickle DA et al. Optimal delivery of blood cardioplegia. Circulation 1991;84(Suppl 3):38M.