Are temperatures attained by donor hearts during transport too cold?

J THoRAc CARDIOVASC SURG 1989;98:517-22

Are temperatures attained by donor hearts during transport too cold? Excessive myocardial cooling may have detrimental effects on donor heart integrity. This study assessed the standard technique for donor myocardial preservation using hearts from seven mongrel dogs (mean weight 192.7 gm), which were arrested, excised, and placed in a cooler containing saline and ice. Temperature probes placed in both the left and right ventricular free walls and the septum revealed that, after cardioplegia, temperatures feU to 10.3°, 7.5°, and 7.6° C, respectively. Temperature decreased to below 1° C after 75, 75, and 60 minutes for the left ventricle, right ventricle, and septum, respectively, independent of the size of the heart (range = 104 to 322 gm), After 4 hours of cooling, temperature was below 0° C throughout the myocardium. Examination with an electron microscope showed similar serial changes over 4 hours in aU hearts, including moderate-to-severe cytoplasmic and nuclear sweUing and mitochondrial calcium deposits. CeU membranes remained intact, which suggests that the damage was not irreversible. We conclude that current donor heart preservation techniques may result in unacceptably low myocardial temperatures that cause reversible myocardial injury.

P. J. Hendry, MD, FRCSC,a V. M. Walley, MD, FRCPC,b A. Koshal, MD, FRCSC,a R. G. Masters, MD, FRCSC,a and W. J. Keon, MD, FRCSC,a Ottawa, Ontario, Canada

Athough cardiac transplantation has gained wideacceptance as treatment for end-stage heart failure, the techniques used for donor heart harvesting and transplantationhave not changed appreciably since 1968. Distantly procured hearts are usually arrested with a cardioplegic solution and placed on ice for transport to the transplant center. It is generally agreed that a temperature of 4 C should be used for the cardioplegicsolution. The same temperature is believed to be achieved by myocardiumplacedon ice whilein a cooler. Despite the simplicity of the technique, the question arises as to the exact temperature that the muscle reaches during its transportation. Most transplant centers report similar transport techniques, but temperature is not monitored generally. In our early experience, we noted that the epicardial fat

was often frozen after transport, which gave rise to concerns about the rest of the heart. These experiments were designed to assess the rate of cooling and final temperature attained by donor hearts prepared in a standard fashion for transport. Histologic changes were also assessed during cooling to identify injury during the transport period.

0

From theDepartment ofCardiac Surgery. University ofOttawa Heart Institute: and Department of Pathology. Ottawa Civic Hospital," Ottawa. Ontario. Canada. Received for publication July 2~. 198~. Accepted for publication Feb. 13. 1989. Address for reprints: P. J. Hendry. MD, Lniversity of Ottawa Heart Institute, OttawaCivic Hospital. 1053 Carling Ave.. Ottawa.Ontario KIJ 4E9. Canada. 12/1/11840

Methods and materials Mongrel dogs weighing between 11.5 and 48.5 kg were anesthetized with intravenous pentobarbital (Somnotol, 26 mg/kg), intubated, and the lungs ventilated with a tidal volume of 12 to 15 ml/kg (approved by the University of Ottawa Animal Experimentation Committee). Pancuronium (0.1 rug/kg) was given intravenously for muscle relaxation. Central venous and mean arterial pressures were monitored as were heart rate and electrocardiogram. Left thoracotomy was performed and cardiectomy was done by the technique described by Copeland.' The aorta, superior vena cava, and inferior vena cava were isolated. The superior vena cava was ligated. The aorta was clamped and 500 ml of 51. Thomas' Hospital cardioplegic solution was infused into the aorta proximal to the clamp. The inferior vena cava and right pulmonary veins were opened for drainage, and the pericardium was irrigated with cold saline at 4° C. Once the heart was arrested, cardiectomy was completed by dividing the cavae, pulmonary veins, aorta, and pulmonary artery. The heart was then placed in a plastic bag filledwith cold saline. At this point, the probes of isolated rapid-response

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Surgery

10.0

8.0

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.

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0 60 -2.0

120

180

240

Time (mins.)

Fig. 1. LV free wall temperature plotted against time (mean ± standard error of the mean).

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Fig. 2. RV free wall temperature plotted against time (mean ± standard error of the mean).

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Fig. 3. Septal temperature plotted against time (mean ± standard error of the mean).

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Fig. 4. Electron micrographs of (A) control section of LV at 0 hours, compared with (B) at 2 hours after cold storage. Intracellular edema is obvious, as are nuclear swelling and mitochondrial changes. (Original magnification X 7000.)

minithermometers (model 8517-12, Cole-Parmer Instrument Co., Chicago, Ill.) were placed through stab wounds in the free wallsof the right (R V) and left (LV) ventricles and septum and secured in place with purse-string sutures. The first bag was placed in a second bag containing cold saline, which was placed on ice in a cooler. A temperature probe was placed in the ice under the bag containing the heart. Temperatures were monitored for 4 hours. Full-thickness biopsy specimens of the RV and LV free walls were obtained immediately after cardiectomy, at 2 hours, and

at 4 hours. They were immediately placed in Universal fixative (4% formalin/ I% glutaraldehyde). Full-width cross sections were processed in standard fashion and stained with hematoxylin phloxine saffron for examination with a light microscope. Samples of the midmyocardium for examination with an electron microscope were washed in sodium cacodylate buffer (0.1 rnol/L) and osmicated in I % osmium tetroxide in sodium cacodylate (0.1 mol/L) with I % potassium ferricyanide. Tissue was stained en bloc with saturated aqueous uranyl acetate and embedded in Epon-Araldite resin. Sections I /Lm thick were

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Table I. Frequency of changes identified with an electron microscope in biopsy specimens obtained at different times from LVs and RVs (n = 7) Time Characteristics of biopsy specimens Intercellular/intracellular edema Mitochondrial changes Changes in nuclei

o hr

:! hr

4 hr

LV

RV

LV

RV

LV

RV

2 (2X.6';;)

5

7 (100';':) 4 (57.1';':) 3 (42.9';':)

7 (100';) 3 (42.9';;) 4 (57.I'lr)

7 ( 100';) 4 (57.1~; ) 3 (42.9';; )

7 (100";) 5 (71.4';) 4 (57.1 ';;)

I (14.Y7,)

(71.4'7,) 2 (2X.6f t )

0

0

(0';;)

(0';':)

stained with methylene blue and examined with a light microscope. Thin (500 to 600 A) sections were prepared with a Reichert Ultracut ultramicrotome and diamond knife (ReichertJung, Buffalo, N.Y.), stained with lead citrate, and examined with a Philips 301 electron microscope (Philips Electronic Instruments lnc., Mahwah. N J.) for ultrastructural changes. Statistical analysis was performed by Pearson's correlation coefficient, with p values less than 0.05 being considered statistically significant.

Results

Seven mongrel dogs were used. Their mean weight was 25.8 ± 5.0 kg ( ± standard error of the mean) with a range from 11.5 to 48.5 kg. The excised hearts weighed 192.7 ± 33.2 gm with a range from 103.7 to 322.2 gm. These hearts are somewhat smaller than normal human hearts, which weigh between 250 and 350 gm. After cold cardioplegic arrest, the mean temperatures for the LV, RV, and septum were 10.3 0 ± 1.1 0, 7.5° ± 1.1 ", and 7.6 ° ± 1.0° C, respectively. The ice temperature throughout the experiment was relatively constant at between -0.2° and -0.4° C. The most precipitous fall in temperature occurred in the first 60 minutes of cooling. The LV cooled to 103 ° ± OJ °C, and the R V and septum fell to temperatures of 1.3° ± 0.5 0 and 0.7 0 ± 0.2° C, respectively. Cooling was slower to 4 hours, with myocardium attaining temperatures that were the same as those of the surrounding ice. The rate of change of myocardial temperature with respect to time for the LV, RV, and septum are shown graphically in Figs. 1, 2, and 3, respectively. The weight of the heart did not correlate with the temperature achieved after cold cardioplegic arrest (r = 0.40, P = 0.37). However, there was a positive correlation between heart weight and temperature after 60 minutes (r = 0.76, P = 0.05). Biopsy specimens of myocardium were taken in such a way that immediate endocardial surfaces were absent in about half of the specimens. The only abnormality discernible with a light microscope was vacuolation of the myocytes in the immediate subendocardium. This change

did not have any correlation with the time interval after removal, although it was much more prominent in the RV sections. Examination of the thick electron microscopy sections revealed a vague tendency, difficult to quantify, for the myocytes to appear slightly edematous, with accentuated cytoplasmic granularity as the time after removal increased. This impression was not appreciably different when the LV was compared with the RV. Ultrastructural examination of the myocardium identified some trends (Fig. 4, A and B and Fig. 5). When compared with the control specimens taken at 0 hours, the biopsy specimens at 2 and 4 hours showed increasing degrees of intracellular and intercellular edema with some associated disruption of myofilaments. The degree of this change became progressively more marked at the 2-hour and 4-hour times. Mitochondria were contracted in some cases and swollen in others. Nuclei tended to become swollen and cleared of chromatin (Table I). Capillaries in the interstitium showed a tendency for endothelial swelling. In no case were irreversible degenerative changes apparent. Discussion

Although variations exist between transplant centers, donor cardiectomy and transport techniques are similar. 2•s The heart is arrested with a cold cardioplegic solution and placed in bags filled with cold saline. The bags are then placed in an ice chest, and the temperature is thought to be maintained at 4° C. Temperature monitoring does not seem to be a concern expressed by most centers, though most transplant surgeons have opened their ice chests after transport and found organs with evidence of at least surface freezing. It would seem intuitive that any isolated tissue exposed to 0° C temperatures would eventually freeze and not maintain 4 ° C temperature. The results of these experiments support this state-

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Fig. 5. Electron micrographof detail in Fig. 4, B. Intracellular edema is obvious, as are degenerative cytoplasmic changes, including mitochondrial abnormalities with amorphousdensities. (Original magnification X 11,200.)

ment. Once isolated from the warming influences of operating room lights, handling, and collateral blood flow, thedonor heart cools precipitously. When it is placed in saline and ice,the temperature decrease is more dramatic, with cooling to less than 1° C in just over 60 minutes. After3 hours,the myocardial temperature may decrease to below 0° C. Initially, myocardial temperature decreased at a rate that was independent of heart weight. All parts of the heart were cooled uniformly by coronary perfusion with acold cardioplegic solution. However, after 60 minutes of cooling in isolation, the temperature attained by the myocardium correlated well with the weight of the heart. In isolation, the heart acted as a solid mass, which resulted in longer cooling times for larger hearts. This essentially demonstrates the difference in cooling achieved by perfusion and topical techniques. An abundance of literature exists concerning techniques for myocardial protection during cardiac operations. The donor heart, however, presents different conditions inthat the heart is isolated from warming influences and is ischemic for 4 to 6 hours. This study does not attempt to identify the best temperature for myocardial preservation but clarifies what happens to temperature with currently practiced donor heart procurement techniques. The ideal temperature for graft transport is controversial, with numerous groups advocating tempera-

tures from 0° C to 15° C on the basis of differing results. Although 0° C has been shown experimentally by some to allow good return of function.v- 7 others have shown the opposite.f At 4 ° C, function 7.9 and adenosine triphosphate stores are well preserved.'? However, cellular ultrastructure seems to be best maintained at 14 ° C. l l , 12 Recent experiments with human right atrial trabeculae suggest that a 10° to 12° C temperature is optimal.f Swanson, Dufek, and Kahn 13 however, could not identify any major differences in return of function of hearts preserved at 4° and 15° C. Excessive cooling (i.e., less than 4 ° C) should be avoided inasmuch as resultant cellular derangements lead to reperfusion injury." To achieve this end, we are now monitoring temperature with a sterile thermometer placed in the second bag of saline. This allows for good visibility to read the thermometer while at the same time giving a relatively reliable myocardial temperature. We often have to remove the heart from the ice and place it on top of the cooler as temperatures reach the 4 ° C level. Since adopting this practice, we have not had any frozen hearts. The benefits of carefully maintaining cooling temperatures are harder to prove. In these experiments, ultrastructural changes appeared that are associated with cooling injury. These include intracellular edema, mitochondrial swelling, and nuclear chromatin changes. These changes worsened with time. However, the same changes

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may be a result of ischemic damage, and it would be difficult to differentiate accurately between the two injuries. No changes were considered to be irreversible despite cooling temperatures near 0° C. The Stanford group has found that changes occur in myocardial ultrastructure after 3 hours of donor heart ischemia, and these changes tend to worsen after reperfusion.P Emery and associates- noted that the longterm survival rates of recipients of hearts from distant donors were poorer than the survival rates of patients who received local hearts. This observation may be explained by greater ischemic times, but excessive cooling in an ice chest may be a contributing factor. Lurie and associates 16 showed that rat hearts exposed to 2 ° to 4 ° C solutions for more than 4 hours had marked transmural fibrosis. Clearly, more study is needed in determining a relationship between donor heart function and cooling temperature. At present, most groups report excellent results from transplantation. A normal heart from a young donor would likely function well initially despite ischemic or cold injury, or both. Our goal should be to preserve the heart as well as possible. Intratransport myocardial temperature monitoring, as suggested, or the development of a small refrigeration device with a constant temperature may be of use. Current methods of transporting donor hearts result in excessively cold temperatures and may result in myocardial injury. Further investigation is currently being undertaken to identify the ideal method for donor heart preservation. We wish to thank Mrs. Misa Bose-Davies, the Experimental Surgery Department at the University of Ottawa Health Science Center, and the histology and electron microscopy labora tories ofthe Division of Anatomical Pathology a t the Ottawa Civic Hospital for their technical assistance. REFERENCES 1. Copeland JG. Heart transplantation. In: Modern technics in surgery: cardiac thoracic surgery. Mount Kisco, New York: Futura, 1984.

Thoracic and Cardiovascular Surgery

·2. Watson DC, Reitz BA, Baumgartner W A, et al. Distant heart procurement for transplantation. Surgery 1979;86: 56-9. 3. Emery RW, Cork RC, Levinson MM, et al. The cardiac donor: a six-year experience. Ann Thorac Surg 1986;41: 356-62. 4. Griffith BP, Trento A, Kormos RL, Hardesty RL, Bahnson HT. Cardiac transplantation. Ann Surg 1986;204:308-14. 5. Slater AD, Klein JB, Gray LA. Clinical orthotopic cardiac transplantation. Am J Surg 1987; 153:582-93. 6. Kohno H, Shiki K, Ueno Y, Tokunaga K. Cold storage of the rat heart for transplantation. J THORAC CARDIOVASC SURG 1987;93:86-94. 7. Shragge BW, Digerness SB, Blackstone EH. Complete recovery of the heart following exposure to profound hypothermia. J THORAC CARD10VASC SURG 1981;81:455-8. 8. Keon WJ, Hendry PJ, Taichman GC, Mainwood GW. Cardiac transplantation: the ideal myocardial temperature for graft transport. Ann Thorac Surg 1988;46:337-41. 9. Guerraty A, Alivizatos P, Warner M, Hess M, Allen L. Lower R. Successful orthotopic canine heart transplantation after 24 hours of in vitro preservation. J THORAC CARDIOVASC SURG 1981;82:531-7. 10. Kao RL, Conti VR, Williams EH. Effect of temperature during potassium arrest on myocardial metabolism and function. J THORAC CARDIOVASC SURG 1982;84:243-9. II. Balderman SC, Binette P, Chan A WK, Gage AA. The optimal temperature for preservation of the myocardium during global ischemia. Ann Thorac Surg 1983;35:605-14. 12. Engedal H, Skagseth E, Saetersdal S, Myklebust R. Cardiac hypothermia evaluated by ultrastructural studies in man. J THoRAc CARDIOVASC SURG 1978;75:548-54. 13. Swanson DK, Dufek J, Kahn DR. Improved myocardial preservation at 4 0 C. Ann Thorac Surg 1980;30:519-26. 14. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:167-208. 15. Billingham ME, Baumgartner WA, Watson DC, et al. Distant heart procurement for human transplantation: ultrastructural studies. Circulation 1980;62(Pt 2):111-9. 16. Lurie KG, Billingham ME, Masek MA, et al. Ultrastructural and functional studies on prolonged myocardial preservation in an experimental heart transplant model. J THoRAC CARDIOVASC SURG 1982;84: 122-9.