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Collins’ solution for cold storage of the heart for transplantation must be reversed with cardioplegic solution before reperfusion
Collins' solution for cold storage of the heart for transplantation must be reversed with cardioplegic solution before reperfusion Afunctional and metabolic study in the rat heart The following hypotheses were tested using an isolated perfused working rat heart model: (1) Collins' solution for cold storage of the heart is harmful for the heart during reperfusion; (2) a "reverse" of the intracellular-type Collins' solution with an extracellular-type cardioplegic solution before reperfusion is able to prevent this disadvantage of Collins' solution. The following two major groups (I and II) and five subgroups (-a to -e) in each group were prepared. In group I (reversed group); the hearts were initially stored in Collins' solution but were reversed by a l-minute flush with cardioplegic solution followed by storage in cardioplegic solution for the last 1 to 180 minutes of the total 3-hour storage, that is, groups I-a (reversed for 1 minute), I-b (10 minutes), I-c (30 minutes), I-d (90 minutes), and I-e (180 minutes). In group II (nonreversed control group); the hearts were stored in Collins' solution throughout 3 hours and were also divided into five subgroups of groups II-a, II-b, II-c, II-d, and II-e in which only a I-minute flush with Collins' solution was performed at the point corresponding to group I. The coronary flow in any of group II showed a marked decrease during the early reperfusion period. In group I, however, the coronary flow increased significantly in proportion to the duration of the reversing phase. The recovery of the aortic flow and the cardiac output in group I showed a bell-shaped pattern in relation to the duration of the reversing phase, reaching their peak values when reversed for 30 minutes (group I-c). The prolonged reverse (180 minutes) resulted in a deterioration of functional recovery associated with a poorer preservation of high-energy phosphates and a larger enzyme leakage. These results suggest that the beneficial effects of intracellular-type Collins' solution for cold storage of the heart were further improved by reversing Collins' solution with the extracellular-type cardioplegic solution for the last 30 minutes of the 3-hour cold storage because the disadvantageous vasoconstriction due to Collins' solution during reperfusion was successfully prevented by the replacement of intravascular and extravascular Collins' solution with cardioplegic solution before the reperfusion. (J THoRAc CARDIOVASC SURG 1992;104:1572-81)
Yoshihiro Toshima, MD, Hiroyuki Kohno, MD, Kohji Matsuzaki, MD, Atsuo Mitani, MD, Hisanori Mayumi, MD, Hisataka Yasui, MD, and Kouichi Tokunaga, MD, Fukuoka, Japan
From the Division of Cardiovascular Surgery, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Supported by a Grant-in-Aid for the Encouragement of Young Scientists (63771030) from the Ministry of Education, Science and Culture, Japan. Received for publication Sept. 23, 1991. Accepted for publication June II, 1992. Address for reprints: Yoshihiro Toshima, MD, Division of Cardiovascular Surgery, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.
12/1/40172
1572
h e principle of hypothermic preservation of the heart for transplantation with an intracellular-type solution is to preserve the myocardial viability not only by reducing the myocardial energy requirements I but also by maintaining the intracellular ionic integrity.? On the other hand, an intracellular-type solution also causes some harmful electrophysiologic actions, such as high-potassium contracture.' low-sodium contracture," and calcium paradox' although deep hypothermia may somewhat ameliorate these disadvantages of an intracellular-type solution.v" Kohno and colleagues? have previously reported that two types of solution should be used for
Volume 104 Number 6 December 1992
Collins' solution must be reversed before reperfusion
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optimal heart preservation: the extracellular-type solution (Kyushu University cardioplegic solution) to arrest the heart, and the intracellular-type solution (Collins' solution) to store the heart. When the intracellular-type solution was used to arrest the heart, a harmful vasoconstriction occurred, probably because of the high potassium and the low sodium levels of Collins' solution." Recently, we have further analyzed Kohno's method of cold storage, focusing on the recovery phase of the heart during reperfusion.f The results of our study showed that the hearts that were arrested with cardioplegic solution, flushed with and stored in Collins' solution, showed a much better functional and metabolic recovery than the hearts that were arrested with, flushed with, and stored in cardioplegic solution alone.f The coronary vascular resistance during the early reperfusion period, however, was remarkably higher in the former hearts. This probable disadvantage of the intracellular-type solution may be explained by the similar mechanisms that cause severe vasoconstriction during the arrest-inducing period." If so, the use of an extracellular-type "reverse solution" before the reperfusion might be able to prevent the harmful effects of the intracellular-type solution during reperfusion, as did the use of an "arrest solution,"? and may further improve the preservation of the heart. The aim of the present study was to elucidate the effectiveness of reversing Collins' solution with cardioplegic solution before reperfusion in the isolated rat heart arrested with cardioplegic solution and flushed with and stored in Collins' solution for 3 hours at 4 0 C. Moreover, an optimal duration of this reverse to best preserve myocardial function was also determined.
Materials and methods Experimental design. Seventy-eighthearts from adult male WKA rats (from 280 to 400 gm) were used. All the animals received humane care in compliance with the "Principles of
Table I. Composition of the solutions KHB (mmolfl.i Na+ K+ CaH MgH
144
ClP0 42-
127
soi-
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Glucose Osmolarity pH
5.9 2.5 1.2 1.2 1.2 25
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KHB, Krebs-Henseleitbicarbonate buffer solution; CP, Kyushu University cardioplegicsolution;C, Collins' solution; EDTA, ethylenediaminetetraacetic acid.
Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23,revised 1978).The preparation of the isolatedworkingrat heart modelwas performed as described previously."? After a control perfusion with the modified Krebs-Henseleit bicarbonate buffer solution (Table I), the control values of left ventricular function were recorded. Then the heart was arrested with a I-minute infusion of cold cardioplegic solution (Kyushu University cardioplegic solution'"; see Table I). Subsequently, cold Collins' solution (see Table I) was flushed for I minute, and the heart was inducted into 3 hours of cold storage with Collins' solution (Fig. 1). This method of cold storage as reported by Kohnoand colleagues7 is the general control of this study. The hearts were divided into two major groups (Fig. 2). Group I (reversedgroup). The heart was initially stored in Collins' solution and, at a certain point of the 3 hours of cold storage, the heart was flushed with cardioplegic solution for I minute and stored in cardioplegic solution for the remaining period. Group II (nonreversed control group). The heart was stored
I 5 74
The Journal of Thoracic and Cardiovascular Surgery
Toshima et al.
Control Perfusion
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Fig. 2. Experimental groups. All the hearts were arrested with the cardioplegic solution and flushed with Collins' solution and then inducted into the cold storage for 3 hours principally with Collins' solution. Two major groups (group I, reversed group: group II; nonreversed control group) and five subgroups in each were prepared according to the intervening procedures during the storage.
in Collins' solution throughout 3 hours of cold storage. To clarify the optimal duration of the reversed phase, group I was divided into five subgroups: l-a was reversed with cardioplegic solution for 1 minute; I-b was reversed with cardioplegic solution for 10 minutes; l-c was reversed with cardioplegic solution for 30 minutes; I-d was reversed with cardioplegic solution for 90 minutes; and I-e was reversed with cardioplegic solution for 180 minutes. Corresponding to these subgroups of group I, group II was also divided into the five subgroups to match the effects of the I-minute flush and its timing; for example, to wash out any toxic metabolites or to deliver any substrate, or both, I 1 during simple storage: II-a was flushed with Collins' solution for 1 minute just before reperfusion; II-b was flushed with Collins' solution for I minute at 10 minutes before reperfusion; II-c was flushed with Collins' solution for 1 minute at 30 minutes before
reperfusion; II-d was flushed with Collins' solution for I minute at 90 minutes before reperfusion; and II-e was flushed with Collins' solution for 1 minute at 180 minutes before reperfusion. All hearts in these subgroups of group II were stored in Collins' solution during the remaining ischemic time after the flush with Collins' solution. Reperfusion was initiated at 35° C at a pressure of 80 cm H 20 . During the initial 15 minutes of reperfusion, the hearts were perfused in the Langendorff mode, and coronary effluent was serially measured in volume for evaluation of coronary vascular resistance and was collected for creatine kinase determination. All the hearts were spontaneously defibrillated within 15 minutes of reperfusion. The heart was then converted to working perfusion, and the recovery of cardiac function was recorded at 30 minutes of reperfusion (see Fig. 1).
Volume 104
Collins' solution must be reversed before reperfusion
Number 6 December 1992
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Fig. 3. Serial changes of coronary flow rate (CF) during the early reperfusion period in the hearts in group I-e (closed circle) (totally reversed with the cardioplegic solution for 180 minutes) and those in group II-e (stored in Collins' solution for 180 minutes without any intervention). A marked decrement of CF was observed in the hearts stored in Collins' solution (group II-e, open circle). N = 6 in both of the two groups. SEM, Standard error of the mean.
Perfusion and storage apparatus. Our perfusion apparatus has already been described previously in detail" 9 and is similar to that of Neely and colleagues.'? In the present experiments, Langendorff perfusion was performed at a pressure of 80 em H20, and the working perfusion was performed at a left atrial pressure of 18 cm H20 with the Krebs-Henseleit bicarbonate buffer solution gassed with 95% oxygen and 5% carbon dioxide and maintained at 35° C. The infusion of cardioplegic solution and Collins' solution was accomplished at a constant flow rate of 6.5 ml /rnin with a syringe infusion pump (Compact Infusion Pump Model 975, Harvard Apparatus, South Natick, Mass.). The hearts were stored in a fluid-filled glass that was immersed in a refrigerating bath (RTE-8, Neslab Instruments Inc., Portsmouth, N.H.) while strictly maintaining the temperature at 4.0° ± 0.5° C. Measurement of left ventricular function. Aortic pressure (AP) was monitored via a side arm of the aortic cannula with a pressure transducer (Statham model P50, Gould Inc., Oxnard, Calif.). Heart rate (HR) was obtained from AP with amplifier units (System 365-12, NEC San-ei, Tokyo, Japan). Aortic and coronary flow rates (AF and CF) were measured by timed collection. Cardiac output (CO) was derived from the sum of AF and CF. The indices of functional recovery were expressed as percentages of the individual prearrest control values. Creatine kinase analysis. Coronary effluent during the first IS minutes of reperfusion was collected in an ice-cooled beaker and stored at -70 0 C until analyzed. The enzyme activity was measured with a kit (Worthington, Biochemical Corp., Freehold, N.J.) and a spectrophotometer (Hitachi 200-20, Hitachi Inc., Tokyo, Japanj.l'' The total amount of creatine kinase leakage was expressed as international units per heart. Myocardial metabolite analysis. For the analysis of myocardial metabolites, the hearts were freeze clamped with a Wollenberg clamp that was precooled in liquid nitrogen. Six hearts were sampled for preischemic control values at the end of the control perfusion. At the end of storage, every six hearts in groups l-c, II-c, I-e, and II-e, in which clear differences in the functional recovery had been observed, were sampled. The frozen samples were stored at 70° C and then used to determine
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Fig. 4. The effect of the timing of the I-minute flush with Collins' solution during cold storage on the recovery of coronary flow rate (CF) during the early reperfusion period in group II. There was no difference in the recovery patterns of CF among the hearts in groups II-a, II-b, II-c, and II-d. These recovery patterns were also similar to that in group II-e. N = 6 in each group. SEM, Standard error of the mean. the tissue contents of adenosine triphosphate, creatine phosphate, and lactate by standard enzymatic methods!" with the spectrophotometer. The values of metabolite contents were expressed as micromoles per gram dry weight. Statistical analysis. Each value was expressed as the mean ± standard deviation in the text. An evaluation of the data corresponding between groups I and II (i.e., I-a versus II-a) was conducted by unpaired Student's t test. A comparison among the five subgroups in group I or group II was conducted by analysis of variance followed by the unpaired Student's t test with the Bonferroni correction." Probability values of less than 0.05 were taken to be statistically significant. Results CF during the early reperfusion period. CF was measured every minute during the initial 15 minutes of reperfusion in six hearts in each group. The hearts in group Ie (in which Collins' solution was reversed with cardioplegic solution right after the storage was initiated and the hearts were stored in cardioplegic for 180 minutes), all showed a remarkable increase of CF, giving a peak level of 153.6% ± 21.4% of the preischemic control value at 5 minutes ofreperfusion (Fig. 3). In contrast, the hearts in Group II-e, which were stored in Collins' solution for 3 hours without any intervention, showed a marked decrease of CF soon after the initiation of reperfusion with a minimum of 55.3% ± 14.2% at 8 minutes of reperfusion. All the hearts in the other subgroups of group II (nonreversed group) showed a similar recovery pattern ofCF (Fig. 4), indicating that the I-minute flush with the storage solution itself and its timing had no influence on the recovery pattern of CF if the storage solution was not reversed. The reverse with cardioplegic solution before the reperfusion brought a significant increase of CF during the early reperfusion period that was generally in proportion to the duration of the revers-
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The Journal of Thoracic and Cardiovascular Surgery
Toshima et al.
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Volume 104 Number 6 December 1992
Collins' solution must be reversed before reperfusion
1 577
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Fig. 7. Percent recovery of aortic flowrate (AF, upper panel) and percent recoveryof coronary flowrate (CF, lower panel) at 30 minutes of reperfusion. The hearts in group I (dotted column) showed a bell-shaped relationship between the recovery of AF and the duration of the reversed phase. Among the hearts in group II (open column), there was no significant difference in the recovery of AF. Patterns of percent recoveryof CF in groups I and II were similar to that of AF. N = 6 in each group. Values are expressed as mean ± standard error of the mean. ANOVA, analysis of variance.
ing phase (Fig. 5). No obvious efficacy of the reverse, however, was found in the hearts reversed for 1 minute or 10 minutes, suggesting that at least 30 minutes is required to replace all of the interstitial fluid sufficiently.
Creatine kinase leakage. The total amount of creatine kinase in the coronary effluent during the first IS minutes of reperfusion was determined in six hearts from each group (Fig. 6). There was no difference among the
The Journal of
Toshima et al.
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Thoracic and Cardiovascular Surgery
( % )
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Fig. 8. Percent recovery of cardiac output (CO) at 30 minutes of reperfusion. Patterns of percent recovery of CO were similar to that of aortic flow rate (AF) in both groups I and II. The best hearts in group I (group l-c) showed significantly better recovery of CO than the best hearts in group II (group II-e). N = 6 in each group. Values were expressed as mean ± standard error of the mean. ANOVA, analysis of variance.
Table II. Myocardial metabolite contents [umolfgm dry weight) at the end of cold storage in the hearts reversed for 30 minutes (I-c), 180 minutes (I-e), and their control hearts (II-e and II-e). ATP Preischemic control heart Group l-c (reversed for 30 min) Group II-c (nonreversed control heart) Group l-e (reversed for 180 min) Group II-e (nonreversed control heart)
25.8 23.1 23.8 17.5 24.1
CrP
± 2.1
± 1.5 ± 1.2 ± 1.0 ± 1.4*
39.4 23.1 25.9 7.8 19.2
± 6.2 ± 5.0 ± 4.4 ± 4.9 ± 4.8*
Lactate 11.2 55.9 50.8 107.2 89.2
± 3.2 ± 3.7 ± 3.5 ± 13.1 ± 9.8t
All values are expressed as mean ± standard deviation. N = 6 in all of the groups. ATP. Adenosine triphosphate; CrP, creatine phosphate.
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hearts in group II (nonreversed group). In contrast, there was a significant difference (p < 0.0 I by analysis of variance) among the hearts in group I (reversed group), indicating a distinctly large quantity of the released creatine kinase in group I-e, in which the heart was stored in cardioplegic solution for 3 hours. When compared with the corresponding hearts of groups I and II, there was no difference except for one difference between groups I-e and II-e. These data clearly demonstrate that Collins' solution is superior to cardioplegic solution in preserving the heart during the cold storage. Moreover, the vasoconstriction during the early reperfusion period itself may not necessarily lead to lethal myocardial cellular injury.
Left ventricular function. Recovery of cardiac function was measured at 30 minutes of reperfusion in six hearts from each group. Recovery of HR and AP was essentially the same among all groups. Recovery of AF (Fig. 7, upper panel) showed no difference among the hearts in group II (nonreversed group). The hearts in group I (reversed group), however, showed a bell-shaped relationship between the recovery of AF and the duration of the reversed phase, showing a best recovery of 73.0% ± 3.1% of the preischemic control value in group I-c, which was statistically significant compared with other subgroups in group I (p < 0.05 by analysis of variance). When a comparison was made between groups I
Volume 104 Number 6 December 1992
Collins' solution must be reversed before reperfusion
and II, group l-c was significantly better than group II-c (73.0% ± 3.1% versus 65.5% ± 2.7%;p < 0.01), whereas group l-e was significantly worse than group II-e (56.6% ± 7.1% versus 68.2% ± 4.8%;p < 0.01). Closely similar findings were seen in recovery of CF (Fig. 7, lower panel) and CO (Fig. 8). It is noteworthy, however, that the final recovery of CF at 30 minutes of reperfusion in group l-e, in which a remarkable increase of CF had been observed during the early reperfusion phase (see Fig. 3), was the worst among all of the groups. Moreover, when a comparison was made between the best of group I (group I-c) and the best of group II (group II-e), recovery of CO in group I-c was significantly better than that in group II-e (p < 0.05; see Fig. 8). Myocardial metabolites. To analyze the metabolic participation in functional recovery, the myocardial metabolite contents were determined at the end of the cold storage in groups l-c, II-c, l-e, and II-e in which clear differences in the recovery of cardiac function had been observed (Table 11). Group l-e, in which the hearts were stored in cardioplegic solution for the entire 180 minutes showed significantly larger decreases of adenosine triphosphate and creatine phosphate and a greater accumulation of lactate compared with Group II-e, corresponding to the worst recovery of cardiac function in group I-e. On the other hand, there was no difference of adenosine triphosphate, creatine phosphate, and lactate values between groups I-c and II-c despite the significant difference in the final functional recovery.
Discussion In our previous study'' we found both beneficial and disadvantageous effects of the intracellular-type solution on myocardial recovery after cold storage; that is, although the hearts stored in the intracellular-type solution showed better metabolic and larger functional recovery than those stored in the extracellular-type solution, they encountered severe vasoconstriction during the early reperfusion phase. This vasoconstriction was considered to be attributed to the ionic composition of the intracellular-type solution with a close relationship to myocardial temperature. Recently, Von Oppell and colleagues 16 reported the effects of normothermic or hypothermic exposure of human venous endothelial cells to various preservation solutions (intracellular-and extracellular-type solutions). They clearly showed that exposure of the endothelial cells to intracellular-type solutions was not more cytotoxic than exposure to an extracellular-type solution in a profoundly hypothermic condition. Such an exposure to intracellular-type solutions was much more cytotoxic than exposure to an extracellular-type solution when the conditions were normothermic. The use of an
I 579
extracellular-type solution at both the beginning and the end of hypothermic heart preservation with an intracellular-type solution appears to be beneficial in the sense that it avoids exposure of the endothelial cells to an intracellular-type solution under normothermic conditions. CF after global ischemia, in general, is affected by myocardial oxygen requirements, 17 tissue adenine nucleosides degraded during ischemia.l? tissue adenosine triphosphate level,18 and myocardial tissue edema.l" In the present study, therefore, the difference in CF between groups I-e (totally stored in cardioplegic solution) and II-e (totally stored in Collins' solution) may be affected by the poorer preservation of the myocardial high-energy phosphates during ischemia in group I-e or the more elevated electromechanical activity during the early reperfusion phase in group I-e (data not shown), which might be closely related to the worst functional recovery in group I-e. On the other hand, the increase of CF in group l-c (reversed for 30 minutes) compared with Group II-c was not associated with either any difference in the myocardial high-energy phosphate contents at the end of the storage or with any difference in the electromechanical activities during the early reperfusion phase as evidenced by the prevalence or duration of tachyarrhythmias and by the HR (data not shown). Thus this increase of the coronary vascular resistance seen in the hearts stored in the intracellular-type solution and not reversed may be attributed solely to the ionic composition of the storage solution that exists in the myocardial tissue at the time of reperfusion. The reason, however, that the increment of CF during the early reperfusion phase by reversing the intracellular-type solution with the extracellular-type solution led to a further improvement of functional recovery is not clear. Lucas and colleagues" reported that the increment of CF during reperfusion by infusing sodium nitroprusside resulted in some increase in the left ventricular rate of pressure rise. In agreement with this result, the recovery of AF and CO was improved in proportion to the increase in the groups reversed for I to 30 minutes in our present system. As reported previously.f the vasoconstriction caused by the intracellular-type solution was associated with a marked decrease of myocardial oxygen uptake. Therefore, we consider that the increase of CF by reversing might also increase myocardial oxygen uptake and lead to further improvement of functional recovery. We have to take into account the possibility, however, that the avoidance of the normothermic contact of myocardium with the intracellular-type solution might also have prevented some harmful effects on myocardial cells themselves, since an infusion of the intracellular-type solution to arrest the heart caused not only severe vasoconstriction but also myocardial cellular injury as evi-
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The Journal of Thoracic and Cardiovascular Surgery
Toshima et at.
denced by a greater amount of creatine kinase leakage during the infusion period." To reverse Collins' solution with cardioplegic solution efficiently, 30 minutes was required before reperfusion while neither I minute nor 10 minutes was sufficient. This time dependency of the reversing efficacy may be attributed to the method we used for the reverse. Alto and Dhalla 22 reported that, to measure the myocardial cation contents in the rat heart, an infusion with 10 ml of the cation-free solution could effectively wash out the perfusion medium and could sufficiently minimize contamination from the extracellular compartment. Based on this finding, the flush with only 6.5 ml of the reverse solution may be too small to replace all of the extracellular space in the present system. Therefore, it may take at least 30 minutes for the passive diffusion of the ions between the residual Collins' solution in the extracellular space and the infused cardioplegic solution in the vessels across the coronary vascular beds to reach their equilibrium states. At present most clinical heart transplant teams use extracellular-type cardioplegic solutions to store donor hearts. The registry of the International Society for Heart and Lung Transplantation.P however, seems to indicate that further improvement in preservation methods is required. Recently, some clinical studiesv'- 25 revealed the superiority of the intracellular-type solutions (Euro-Collins solution and University of Wisconsin solution) in preservation of cardiac performance and electrical stability of the transplanted hearts. We support these trials and expect that some prolongation of safe graft ischemic time will be achieved. Furthermore, our data provide evidence that the use of an extracellular-type "arrest solution" and "reverse solution" is indicated when an intracellular-type solution is used as a "storage solution," although some additives like adenosine might more or less prevent harmful effects of an intracellular-type solution.P Fortunately, the period of 30 minutes before reperfusion that appeared to be optimal for the reversing phase in the present study may correspond to the time required for the anastomosis of the donor heart. Therefore we strongly suggest, based on the results of the present study, that an extracellular-type cardioplegic solution should be aggressively used for intraoperative myocardial preservation, especially after storage with an intracellular-type solution. In conclusion, from the present study we have developed an optimum protocol for the cold storage of the heart for transplantation; that is, the heart is arrested with the cardioplegic solution of extracellular type, flushed with and stored in Collins' solution of intracellular type, and then again reversed with the cardioplegic solution for the last 30 minutes of cold storage. The use of the cardiople-
gic solution before and after storage with Collins' solution is essential to prevent any harmful vasoconstriction caused by Collins' solution. We would like to thank Ms. E. Nakanishi for excellent technical assistance and Associate Professor Brian T. Quinn, at the Institute of Languages and Cultures, Kyushu University, Fukuoka, Japan, for editing the manuscript. REFERENCES 1. Hearse OJ, Braimbridge MY, Jynge P. Hypothermia. In: Protection of the ischemic myocardium: cardioplegia. New York: Raven Press, 1981;167-208. 2. CoIlins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transplantation: initial perfusion and 30 hours' ice storage. Lancet 1969;2:1219-22. 3. Gharagozloo F, Bulkley BH, Hutchins GM, et al. Potassium-induced cardioplegia during normothermic cardiac arrest: morphologic study of the effect of varying concentrations of potassium on myocardial anoxic injury. J THORAC CARDIOVASC SURG 1979;77:602-7. 4. Kinoshita K, Ehara T. Importance of sodium ions in the protective effects of high-potassium, high-glucose solution on electromechanical activities in the guinea-pig myocardium. J Mol Cell Cardiol 1984;16:405-19. 5. Zimmerman ANE, Hulsman We. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966;211:646-7. 6. Baker JE, Bullock GR, Hearse OJ. The temperature dependence of the calcium paradox: enzymatic, functional and morphological correlates of cellular injury. J Mol Cell CardioI1983;15:393-411. 7. Kohno H, Shiki K, Ueno Y, Tokunaga K. Cold storage of the rat heart for transplantation: two types of solution required for optimal preservation. J THORAC CARDIOVASC SURG 1987;93:86-94. 8. Toshima Y, Matsuzaki K, Mitani A, et al. The myocardial recovery mode after cold storage for transplantation with Collins' solution and cardioplegic solution: a functional and metabolic study in the rat heart. J THORAC CARDIOVASC SURG [In press]. 9. Tominaga R, Kawachi Y, Kohda Y, Tokunaga K. Combined effects of temperature, potassium and calcium concentration on cardiac function in the isolated working rat heart. J Jpn Assoc Thorac Surg 1983;31:446-57. 10. Kinoshita K, Oe M, Tokunaga K. Superior protective effect of low-calcium, magnesium-free potassium cardioplegic solution on ischemic myocardium: clinical study in comparison with St. Thomas' Hospital solution. J THORAC CARDIOVASC SURG 1991;101:695-702. 11. Hearse OJ, Braimbridge MY, Jynge P. Principles of formulation and administration. In: Protection of the ischemic myocardium: cardioplegia. New York: Raven Press, 1981;300-26. 12. Neely JR, Liebermeister H, Battersby EJ, Morgan HE.
Volume 104 Number 6
December 1992
13.
14. 15. 16.
17. 18.
19.
20.
Collins' solution must be reversed before reperfusion
Effect of pressure development on oxygen consumption by isolated rat heart. Am J PhysioI1967;212:804-14. Oliver IT. A spectrophotometric method for the determination on creatine phosphokinase myokinase. Biochem J 1955;61:116-22. Bergmeyer HU, ed. Methods of enzymatic analysis. New York: Academic Press, 1974. Glantz SA. Primer of biostatistics. New York: McGrawHill, 1987. Von Oppell UO, Pfeiffer S, Preiss P, Dunne T, Zilla P, Reichart B. Endothelial cell toxicity of solid-organ preservation solutions. Ann Thorac Surg 1990;50:902-10. Gorlin R. Regulation of coronary blood flow. Br Heart J 1971;33(suppl):9-14. Aversano T, Ouyang P, Silverman H. Blockade of the ATP-sensitive potassium channel modulates reactive hyperemia in the canine coronary circulation. Circ Res 1991; 69:618-22. Foglia RP, Steed DL, Follette DM, DeLand E, Buckberg GD. Iatrogenic myocardial edema with potassium cardioplegia. J THORAC CARDIOVASC SURG 1979;78:217-22. Lucas SK, Kanter KR, SchaffHV, Elmer EB, Glower DD,
21.
22.
23.
24.
25.
1 58 1
Gardner TJ. Reduced oxygen extraction during reperfusion: a consequence of global ischemic arrest. J Surg Res 1980;28:434-41. Kohno H, Toshima Y, Nakamura Y, et al. Induction of cardiac arrest before cold storage in Collins' solution: important role of the cardioplegic solution. J Jpn Assoc Thorac Surg 1988;36:2229-32. Alto LE, Dhalla NS. Myocardial cation contents during induction of calcium paradox. Am J Physiol 1979; 237:H713-9. Kriett JM, Kaye MP. The registry of the International Society for Heart and Lung Transplantation: eighth official report-1991. J Heart Lung Transplant 1991;10:491-8. Hausen B, Fieguth HG, Schafers HJ, Winter A, Spring Ead, Haverich A. Distant heart procurement: impacts of storage solution on cardiac performance following transplantation. Transplant Int 1988;1:140-5. Jeevanandam V, Barr ML, Auteri JS, et al. University of Wisconsin solution versus crystalloid cardioplegia for human donor heart preservation: a randomized blinded prospective clinical trial. J THORAC CARDIOVASC SURG 1992;103:194-9.
Yoshihiro Toshima, MD, Hiroyuki Kohno, MD, Kohji Matsuzaki, MD, Atsuo Mitani, MD, Hisanori Mayumi, MD, Hisataka Yasui, MD, and Kouichi Tokunaga, MD, Fukuoka, Japan
From the Division of Cardiovascular Surgery, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Supported by a Grant-in-Aid for the Encouragement of Young Scientists (63771030) from the Ministry of Education, Science and Culture, Japan. Received for publication Sept. 23, 1991. Accepted for publication June II, 1992. Address for reprints: Yoshihiro Toshima, MD, Division of Cardiovascular Surgery, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.
12/1/40172
1572
h e principle of hypothermic preservation of the heart for transplantation with an intracellular-type solution is to preserve the myocardial viability not only by reducing the myocardial energy requirements I but also by maintaining the intracellular ionic integrity.? On the other hand, an intracellular-type solution also causes some harmful electrophysiologic actions, such as high-potassium contracture.' low-sodium contracture," and calcium paradox' although deep hypothermia may somewhat ameliorate these disadvantages of an intracellular-type solution.v" Kohno and colleagues? have previously reported that two types of solution should be used for
Volume 104 Number 6 December 1992
Collins' solution must be reversed before reperfusion
'"
Control Perfusion
'5>
35°C
'5
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L
W
a o
10min
10min
~
Function Coronary Flow" Heart Rate Aortic: Row Cardiac Output
Metabolites
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Q)
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!
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Fig. 1. Experimental protocol.
optimal heart preservation: the extracellular-type solution (Kyushu University cardioplegic solution) to arrest the heart, and the intracellular-type solution (Collins' solution) to store the heart. When the intracellular-type solution was used to arrest the heart, a harmful vasoconstriction occurred, probably because of the high potassium and the low sodium levels of Collins' solution." Recently, we have further analyzed Kohno's method of cold storage, focusing on the recovery phase of the heart during reperfusion.f The results of our study showed that the hearts that were arrested with cardioplegic solution, flushed with and stored in Collins' solution, showed a much better functional and metabolic recovery than the hearts that were arrested with, flushed with, and stored in cardioplegic solution alone.f The coronary vascular resistance during the early reperfusion period, however, was remarkably higher in the former hearts. This probable disadvantage of the intracellular-type solution may be explained by the similar mechanisms that cause severe vasoconstriction during the arrest-inducing period." If so, the use of an extracellular-type "reverse solution" before the reperfusion might be able to prevent the harmful effects of the intracellular-type solution during reperfusion, as did the use of an "arrest solution,"? and may further improve the preservation of the heart. The aim of the present study was to elucidate the effectiveness of reversing Collins' solution with cardioplegic solution before reperfusion in the isolated rat heart arrested with cardioplegic solution and flushed with and stored in Collins' solution for 3 hours at 4 0 C. Moreover, an optimal duration of this reverse to best preserve myocardial function was also determined.
Materials and methods Experimental design. Seventy-eighthearts from adult male WKA rats (from 280 to 400 gm) were used. All the animals received humane care in compliance with the "Principles of
Table I. Composition of the solutions KHB (mmolfl.i Na+ K+ CaH MgH
144
ClP0 42-
127
soi-
HC03 EDTACa-2Na
Glucose Osmolarity pH
5.9 2.5 1.2 1.2 1.2 25
0.5 11 320 rrrOsm/L 7.4 at 35° C
CP
C
(mmol/L)
(mmol/L)
87 20 0.1
10 117
o
o 3
97
15
10
102 3 10
139
139
o o
o
340 rrrOsm/L 7.8 at 4° C
o
300 mOsm/L 7.4 at 4° C
KHB, Krebs-Henseleitbicarbonate buffer solution; CP, Kyushu University cardioplegicsolution;C, Collins' solution; EDTA, ethylenediaminetetraacetic acid.
Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23,revised 1978).The preparation of the isolatedworkingrat heart modelwas performed as described previously."? After a control perfusion with the modified Krebs-Henseleit bicarbonate buffer solution (Table I), the control values of left ventricular function were recorded. Then the heart was arrested with a I-minute infusion of cold cardioplegic solution (Kyushu University cardioplegic solution'"; see Table I). Subsequently, cold Collins' solution (see Table I) was flushed for I minute, and the heart was inducted into 3 hours of cold storage with Collins' solution (Fig. 1). This method of cold storage as reported by Kohnoand colleagues7 is the general control of this study. The hearts were divided into two major groups (Fig. 2). Group I (reversedgroup). The heart was initially stored in Collins' solution and, at a certain point of the 3 hours of cold storage, the heart was flushed with cardioplegic solution for I minute and stored in cardioplegic solution for the remaining period. Group II (nonreversed control group). The heart was stored
I 5 74
The Journal of Thoracic and Cardiovascular Surgery
Toshima et al.
Control Perfusion
4"C Cold Storage
a.. o o
Total
Group I : Reversed Group I-a
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180min
( n=6 in each group) Reversed with CP for 1 min Reversed with CP for 10 min Reversed with CP for 30 min Reversed with CP for 90 min Reversed with CP for 180 min
C
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Fig. 2. Experimental groups. All the hearts were arrested with the cardioplegic solution and flushed with Collins' solution and then inducted into the cold storage for 3 hours principally with Collins' solution. Two major groups (group I, reversed group: group II; nonreversed control group) and five subgroups in each were prepared according to the intervening procedures during the storage.
in Collins' solution throughout 3 hours of cold storage. To clarify the optimal duration of the reversed phase, group I was divided into five subgroups: l-a was reversed with cardioplegic solution for 1 minute; I-b was reversed with cardioplegic solution for 10 minutes; l-c was reversed with cardioplegic solution for 30 minutes; I-d was reversed with cardioplegic solution for 90 minutes; and I-e was reversed with cardioplegic solution for 180 minutes. Corresponding to these subgroups of group I, group II was also divided into the five subgroups to match the effects of the I-minute flush and its timing; for example, to wash out any toxic metabolites or to deliver any substrate, or both, I 1 during simple storage: II-a was flushed with Collins' solution for 1 minute just before reperfusion; II-b was flushed with Collins' solution for I minute at 10 minutes before reperfusion; II-c was flushed with Collins' solution for 1 minute at 30 minutes before
reperfusion; II-d was flushed with Collins' solution for I minute at 90 minutes before reperfusion; and II-e was flushed with Collins' solution for 1 minute at 180 minutes before reperfusion. All hearts in these subgroups of group II were stored in Collins' solution during the remaining ischemic time after the flush with Collins' solution. Reperfusion was initiated at 35° C at a pressure of 80 cm H 20 . During the initial 15 minutes of reperfusion, the hearts were perfused in the Langendorff mode, and coronary effluent was serially measured in volume for evaluation of coronary vascular resistance and was collected for creatine kinase determination. All the hearts were spontaneously defibrillated within 15 minutes of reperfusion. The heart was then converted to working perfusion, and the recovery of cardiac function was recorded at 30 minutes of reperfusion (see Fig. 1).
Volume 104
Collins' solution must be reversed before reperfusion
Number 6 December 1992
~
ii: ~
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Fig. 3. Serial changes of coronary flow rate (CF) during the early reperfusion period in the hearts in group I-e (closed circle) (totally reversed with the cardioplegic solution for 180 minutes) and those in group II-e (stored in Collins' solution for 180 minutes without any intervention). A marked decrement of CF was observed in the hearts stored in Collins' solution (group II-e, open circle). N = 6 in both of the two groups. SEM, Standard error of the mean.
Perfusion and storage apparatus. Our perfusion apparatus has already been described previously in detail" 9 and is similar to that of Neely and colleagues.'? In the present experiments, Langendorff perfusion was performed at a pressure of 80 em H20, and the working perfusion was performed at a left atrial pressure of 18 cm H20 with the Krebs-Henseleit bicarbonate buffer solution gassed with 95% oxygen and 5% carbon dioxide and maintained at 35° C. The infusion of cardioplegic solution and Collins' solution was accomplished at a constant flow rate of 6.5 ml /rnin with a syringe infusion pump (Compact Infusion Pump Model 975, Harvard Apparatus, South Natick, Mass.). The hearts were stored in a fluid-filled glass that was immersed in a refrigerating bath (RTE-8, Neslab Instruments Inc., Portsmouth, N.H.) while strictly maintaining the temperature at 4.0° ± 0.5° C. Measurement of left ventricular function. Aortic pressure (AP) was monitored via a side arm of the aortic cannula with a pressure transducer (Statham model P50, Gould Inc., Oxnard, Calif.). Heart rate (HR) was obtained from AP with amplifier units (System 365-12, NEC San-ei, Tokyo, Japan). Aortic and coronary flow rates (AF and CF) were measured by timed collection. Cardiac output (CO) was derived from the sum of AF and CF. The indices of functional recovery were expressed as percentages of the individual prearrest control values. Creatine kinase analysis. Coronary effluent during the first IS minutes of reperfusion was collected in an ice-cooled beaker and stored at -70 0 C until analyzed. The enzyme activity was measured with a kit (Worthington, Biochemical Corp., Freehold, N.J.) and a spectrophotometer (Hitachi 200-20, Hitachi Inc., Tokyo, Japanj.l'' The total amount of creatine kinase leakage was expressed as international units per heart. Myocardial metabolite analysis. For the analysis of myocardial metabolites, the hearts were freeze clamped with a Wollenberg clamp that was precooled in liquid nitrogen. Six hearts were sampled for preischemic control values at the end of the control perfusion. At the end of storage, every six hearts in groups l-c, II-c, I-e, and II-e, in which clear differences in the functional recovery had been observed, were sampled. The frozen samples were stored at 70° C and then used to determine
• • • • •
mean ± SEM
II:
If'.
1 575
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15 min
Fig. 4. The effect of the timing of the I-minute flush with Collins' solution during cold storage on the recovery of coronary flow rate (CF) during the early reperfusion period in group II. There was no difference in the recovery patterns of CF among the hearts in groups II-a, II-b, II-c, and II-d. These recovery patterns were also similar to that in group II-e. N = 6 in each group. SEM, Standard error of the mean. the tissue contents of adenosine triphosphate, creatine phosphate, and lactate by standard enzymatic methods!" with the spectrophotometer. The values of metabolite contents were expressed as micromoles per gram dry weight. Statistical analysis. Each value was expressed as the mean ± standard deviation in the text. An evaluation of the data corresponding between groups I and II (i.e., I-a versus II-a) was conducted by unpaired Student's t test. A comparison among the five subgroups in group I or group II was conducted by analysis of variance followed by the unpaired Student's t test with the Bonferroni correction." Probability values of less than 0.05 were taken to be statistically significant. Results CF during the early reperfusion period. CF was measured every minute during the initial 15 minutes of reperfusion in six hearts in each group. The hearts in group Ie (in which Collins' solution was reversed with cardioplegic solution right after the storage was initiated and the hearts were stored in cardioplegic for 180 minutes), all showed a remarkable increase of CF, giving a peak level of 153.6% ± 21.4% of the preischemic control value at 5 minutes ofreperfusion (Fig. 3). In contrast, the hearts in Group II-e, which were stored in Collins' solution for 3 hours without any intervention, showed a marked decrease of CF soon after the initiation of reperfusion with a minimum of 55.3% ± 14.2% at 8 minutes of reperfusion. All the hearts in the other subgroups of group II (nonreversed group) showed a similar recovery pattern ofCF (Fig. 4), indicating that the I-minute flush with the storage solution itself and its timing had no influence on the recovery pattern of CF if the storage solution was not reversed. The reverse with cardioplegic solution before the reperfusion brought a significant increase of CF during the early reperfusion period that was generally in proportion to the duration of the revers-
15 7 6
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The Journal of Thoracic and Cardiovascular Surgery
Toshima et al.
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Fig. 5. A-D The effect of the reverse with the cardioplegic solution and its duration on the recovery of coronary flow rate (CF) during the early reperfusion period. The recovery of CF increased in proportion to the duration of the reversing period. N = 6 in each group. Values are expressed as mean ± standard error of the mean.
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=
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Volume 104 Number 6 December 1992
Collins' solution must be reversed before reperfusion
1 577
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Fig. 7. Percent recovery of aortic flowrate (AF, upper panel) and percent recoveryof coronary flowrate (CF, lower panel) at 30 minutes of reperfusion. The hearts in group I (dotted column) showed a bell-shaped relationship between the recovery of AF and the duration of the reversed phase. Among the hearts in group II (open column), there was no significant difference in the recovery of AF. Patterns of percent recoveryof CF in groups I and II were similar to that of AF. N = 6 in each group. Values are expressed as mean ± standard error of the mean. ANOVA, analysis of variance.
ing phase (Fig. 5). No obvious efficacy of the reverse, however, was found in the hearts reversed for 1 minute or 10 minutes, suggesting that at least 30 minutes is required to replace all of the interstitial fluid sufficiently.
Creatine kinase leakage. The total amount of creatine kinase in the coronary effluent during the first IS minutes of reperfusion was determined in six hearts from each group (Fig. 6). There was no difference among the
The Journal of
Toshima et al.
I 578
Thoracic and Cardiovascular Surgery
( % )
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Fig. 8. Percent recovery of cardiac output (CO) at 30 minutes of reperfusion. Patterns of percent recovery of CO were similar to that of aortic flow rate (AF) in both groups I and II. The best hearts in group I (group l-c) showed significantly better recovery of CO than the best hearts in group II (group II-e). N = 6 in each group. Values were expressed as mean ± standard error of the mean. ANOVA, analysis of variance.
Table II. Myocardial metabolite contents [umolfgm dry weight) at the end of cold storage in the hearts reversed for 30 minutes (I-c), 180 minutes (I-e), and their control hearts (II-e and II-e). ATP Preischemic control heart Group l-c (reversed for 30 min) Group II-c (nonreversed control heart) Group l-e (reversed for 180 min) Group II-e (nonreversed control heart)
25.8 23.1 23.8 17.5 24.1
CrP
± 2.1
± 1.5 ± 1.2 ± 1.0 ± 1.4*
39.4 23.1 25.9 7.8 19.2
± 6.2 ± 5.0 ± 4.4 ± 4.9 ± 4.8*
Lactate 11.2 55.9 50.8 107.2 89.2
± 3.2 ± 3.7 ± 3.5 ± 13.1 ± 9.8t
All values are expressed as mean ± standard deviation. N = 6 in all of the groups. ATP. Adenosine triphosphate; CrP, creatine phosphate.
*p
< 0.01
between groups I and II. Il.tp
< 0.05.
hearts in group II (nonreversed group). In contrast, there was a significant difference (p < 0.0 I by analysis of variance) among the hearts in group I (reversed group), indicating a distinctly large quantity of the released creatine kinase in group I-e, in which the heart was stored in cardioplegic solution for 3 hours. When compared with the corresponding hearts of groups I and II, there was no difference except for one difference between groups I-e and II-e. These data clearly demonstrate that Collins' solution is superior to cardioplegic solution in preserving the heart during the cold storage. Moreover, the vasoconstriction during the early reperfusion period itself may not necessarily lead to lethal myocardial cellular injury.
Left ventricular function. Recovery of cardiac function was measured at 30 minutes of reperfusion in six hearts from each group. Recovery of HR and AP was essentially the same among all groups. Recovery of AF (Fig. 7, upper panel) showed no difference among the hearts in group II (nonreversed group). The hearts in group I (reversed group), however, showed a bell-shaped relationship between the recovery of AF and the duration of the reversed phase, showing a best recovery of 73.0% ± 3.1% of the preischemic control value in group I-c, which was statistically significant compared with other subgroups in group I (p < 0.05 by analysis of variance). When a comparison was made between groups I
Volume 104 Number 6 December 1992
Collins' solution must be reversed before reperfusion
and II, group l-c was significantly better than group II-c (73.0% ± 3.1% versus 65.5% ± 2.7%;p < 0.01), whereas group l-e was significantly worse than group II-e (56.6% ± 7.1% versus 68.2% ± 4.8%;p < 0.01). Closely similar findings were seen in recovery of CF (Fig. 7, lower panel) and CO (Fig. 8). It is noteworthy, however, that the final recovery of CF at 30 minutes of reperfusion in group l-e, in which a remarkable increase of CF had been observed during the early reperfusion phase (see Fig. 3), was the worst among all of the groups. Moreover, when a comparison was made between the best of group I (group I-c) and the best of group II (group II-e), recovery of CO in group I-c was significantly better than that in group II-e (p < 0.05; see Fig. 8). Myocardial metabolites. To analyze the metabolic participation in functional recovery, the myocardial metabolite contents were determined at the end of the cold storage in groups l-c, II-c, l-e, and II-e in which clear differences in the recovery of cardiac function had been observed (Table 11). Group l-e, in which the hearts were stored in cardioplegic solution for the entire 180 minutes showed significantly larger decreases of adenosine triphosphate and creatine phosphate and a greater accumulation of lactate compared with Group II-e, corresponding to the worst recovery of cardiac function in group I-e. On the other hand, there was no difference of adenosine triphosphate, creatine phosphate, and lactate values between groups I-c and II-c despite the significant difference in the final functional recovery.
Discussion In our previous study'' we found both beneficial and disadvantageous effects of the intracellular-type solution on myocardial recovery after cold storage; that is, although the hearts stored in the intracellular-type solution showed better metabolic and larger functional recovery than those stored in the extracellular-type solution, they encountered severe vasoconstriction during the early reperfusion phase. This vasoconstriction was considered to be attributed to the ionic composition of the intracellular-type solution with a close relationship to myocardial temperature. Recently, Von Oppell and colleagues 16 reported the effects of normothermic or hypothermic exposure of human venous endothelial cells to various preservation solutions (intracellular-and extracellular-type solutions). They clearly showed that exposure of the endothelial cells to intracellular-type solutions was not more cytotoxic than exposure to an extracellular-type solution in a profoundly hypothermic condition. Such an exposure to intracellular-type solutions was much more cytotoxic than exposure to an extracellular-type solution when the conditions were normothermic. The use of an
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extracellular-type solution at both the beginning and the end of hypothermic heart preservation with an intracellular-type solution appears to be beneficial in the sense that it avoids exposure of the endothelial cells to an intracellular-type solution under normothermic conditions. CF after global ischemia, in general, is affected by myocardial oxygen requirements, 17 tissue adenine nucleosides degraded during ischemia.l? tissue adenosine triphosphate level,18 and myocardial tissue edema.l" In the present study, therefore, the difference in CF between groups I-e (totally stored in cardioplegic solution) and II-e (totally stored in Collins' solution) may be affected by the poorer preservation of the myocardial high-energy phosphates during ischemia in group I-e or the more elevated electromechanical activity during the early reperfusion phase in group I-e (data not shown), which might be closely related to the worst functional recovery in group I-e. On the other hand, the increase of CF in group l-c (reversed for 30 minutes) compared with Group II-c was not associated with either any difference in the myocardial high-energy phosphate contents at the end of the storage or with any difference in the electromechanical activities during the early reperfusion phase as evidenced by the prevalence or duration of tachyarrhythmias and by the HR (data not shown). Thus this increase of the coronary vascular resistance seen in the hearts stored in the intracellular-type solution and not reversed may be attributed solely to the ionic composition of the storage solution that exists in the myocardial tissue at the time of reperfusion. The reason, however, that the increment of CF during the early reperfusion phase by reversing the intracellular-type solution with the extracellular-type solution led to a further improvement of functional recovery is not clear. Lucas and colleagues" reported that the increment of CF during reperfusion by infusing sodium nitroprusside resulted in some increase in the left ventricular rate of pressure rise. In agreement with this result, the recovery of AF and CO was improved in proportion to the increase in the groups reversed for I to 30 minutes in our present system. As reported previously.f the vasoconstriction caused by the intracellular-type solution was associated with a marked decrease of myocardial oxygen uptake. Therefore, we consider that the increase of CF by reversing might also increase myocardial oxygen uptake and lead to further improvement of functional recovery. We have to take into account the possibility, however, that the avoidance of the normothermic contact of myocardium with the intracellular-type solution might also have prevented some harmful effects on myocardial cells themselves, since an infusion of the intracellular-type solution to arrest the heart caused not only severe vasoconstriction but also myocardial cellular injury as evi-
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The Journal of Thoracic and Cardiovascular Surgery
Toshima et at.
denced by a greater amount of creatine kinase leakage during the infusion period." To reverse Collins' solution with cardioplegic solution efficiently, 30 minutes was required before reperfusion while neither I minute nor 10 minutes was sufficient. This time dependency of the reversing efficacy may be attributed to the method we used for the reverse. Alto and Dhalla 22 reported that, to measure the myocardial cation contents in the rat heart, an infusion with 10 ml of the cation-free solution could effectively wash out the perfusion medium and could sufficiently minimize contamination from the extracellular compartment. Based on this finding, the flush with only 6.5 ml of the reverse solution may be too small to replace all of the extracellular space in the present system. Therefore, it may take at least 30 minutes for the passive diffusion of the ions between the residual Collins' solution in the extracellular space and the infused cardioplegic solution in the vessels across the coronary vascular beds to reach their equilibrium states. At present most clinical heart transplant teams use extracellular-type cardioplegic solutions to store donor hearts. The registry of the International Society for Heart and Lung Transplantation.P however, seems to indicate that further improvement in preservation methods is required. Recently, some clinical studiesv'- 25 revealed the superiority of the intracellular-type solutions (Euro-Collins solution and University of Wisconsin solution) in preservation of cardiac performance and electrical stability of the transplanted hearts. We support these trials and expect that some prolongation of safe graft ischemic time will be achieved. Furthermore, our data provide evidence that the use of an extracellular-type "arrest solution" and "reverse solution" is indicated when an intracellular-type solution is used as a "storage solution," although some additives like adenosine might more or less prevent harmful effects of an intracellular-type solution.P Fortunately, the period of 30 minutes before reperfusion that appeared to be optimal for the reversing phase in the present study may correspond to the time required for the anastomosis of the donor heart. Therefore we strongly suggest, based on the results of the present study, that an extracellular-type cardioplegic solution should be aggressively used for intraoperative myocardial preservation, especially after storage with an intracellular-type solution. In conclusion, from the present study we have developed an optimum protocol for the cold storage of the heart for transplantation; that is, the heart is arrested with the cardioplegic solution of extracellular type, flushed with and stored in Collins' solution of intracellular type, and then again reversed with the cardioplegic solution for the last 30 minutes of cold storage. The use of the cardiople-
gic solution before and after storage with Collins' solution is essential to prevent any harmful vasoconstriction caused by Collins' solution. We would like to thank Ms. E. Nakanishi for excellent technical assistance and Associate Professor Brian T. Quinn, at the Institute of Languages and Cultures, Kyushu University, Fukuoka, Japan, for editing the manuscript. REFERENCES 1. Hearse OJ, Braimbridge MY, Jynge P. Hypothermia. In: Protection of the ischemic myocardium: cardioplegia. New York: Raven Press, 1981;167-208. 2. CoIlins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transplantation: initial perfusion and 30 hours' ice storage. Lancet 1969;2:1219-22. 3. Gharagozloo F, Bulkley BH, Hutchins GM, et al. Potassium-induced cardioplegia during normothermic cardiac arrest: morphologic study of the effect of varying concentrations of potassium on myocardial anoxic injury. J THORAC CARDIOVASC SURG 1979;77:602-7. 4. Kinoshita K, Ehara T. Importance of sodium ions in the protective effects of high-potassium, high-glucose solution on electromechanical activities in the guinea-pig myocardium. J Mol Cell Cardiol 1984;16:405-19. 5. Zimmerman ANE, Hulsman We. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966;211:646-7. 6. Baker JE, Bullock GR, Hearse OJ. The temperature dependence of the calcium paradox: enzymatic, functional and morphological correlates of cellular injury. J Mol Cell CardioI1983;15:393-411. 7. Kohno H, Shiki K, Ueno Y, Tokunaga K. Cold storage of the rat heart for transplantation: two types of solution required for optimal preservation. J THORAC CARDIOVASC SURG 1987;93:86-94. 8. Toshima Y, Matsuzaki K, Mitani A, et al. The myocardial recovery mode after cold storage for transplantation with Collins' solution and cardioplegic solution: a functional and metabolic study in the rat heart. J THORAC CARDIOVASC SURG [In press]. 9. Tominaga R, Kawachi Y, Kohda Y, Tokunaga K. Combined effects of temperature, potassium and calcium concentration on cardiac function in the isolated working rat heart. J Jpn Assoc Thorac Surg 1983;31:446-57. 10. Kinoshita K, Oe M, Tokunaga K. Superior protective effect of low-calcium, magnesium-free potassium cardioplegic solution on ischemic myocardium: clinical study in comparison with St. Thomas' Hospital solution. J THORAC CARDIOVASC SURG 1991;101:695-702. 11. Hearse OJ, Braimbridge MY, Jynge P. Principles of formulation and administration. In: Protection of the ischemic myocardium: cardioplegia. New York: Raven Press, 1981;300-26. 12. Neely JR, Liebermeister H, Battersby EJ, Morgan HE.
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13.
14. 15. 16.
17. 18.
19.
20.
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Effect of pressure development on oxygen consumption by isolated rat heart. Am J PhysioI1967;212:804-14. Oliver IT. A spectrophotometric method for the determination on creatine phosphokinase myokinase. Biochem J 1955;61:116-22. Bergmeyer HU, ed. Methods of enzymatic analysis. New York: Academic Press, 1974. Glantz SA. Primer of biostatistics. New York: McGrawHill, 1987. Von Oppell UO, Pfeiffer S, Preiss P, Dunne T, Zilla P, Reichart B. Endothelial cell toxicity of solid-organ preservation solutions. Ann Thorac Surg 1990;50:902-10. Gorlin R. Regulation of coronary blood flow. Br Heart J 1971;33(suppl):9-14. Aversano T, Ouyang P, Silverman H. Blockade of the ATP-sensitive potassium channel modulates reactive hyperemia in the canine coronary circulation. Circ Res 1991; 69:618-22. Foglia RP, Steed DL, Follette DM, DeLand E, Buckberg GD. Iatrogenic myocardial edema with potassium cardioplegia. J THORAC CARDIOVASC SURG 1979;78:217-22. Lucas SK, Kanter KR, SchaffHV, Elmer EB, Glower DD,
21.
22.
23.
24.
25.
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Gardner TJ. Reduced oxygen extraction during reperfusion: a consequence of global ischemic arrest. J Surg Res 1980;28:434-41. Kohno H, Toshima Y, Nakamura Y, et al. Induction of cardiac arrest before cold storage in Collins' solution: important role of the cardioplegic solution. J Jpn Assoc Thorac Surg 1988;36:2229-32. Alto LE, Dhalla NS. Myocardial cation contents during induction of calcium paradox. Am J Physiol 1979; 237:H713-9. Kriett JM, Kaye MP. The registry of the International Society for Heart and Lung Transplantation: eighth official report-1991. J Heart Lung Transplant 1991;10:491-8. Hausen B, Fieguth HG, Schafers HJ, Winter A, Spring Ead, Haverich A. Distant heart procurement: impacts of storage solution on cardiac performance following transplantation. Transplant Int 1988;1:140-5. Jeevanandam V, Barr ML, Auteri JS, et al. University of Wisconsin solution versus crystalloid cardioplegia for human donor heart preservation: a randomized blinded prospective clinical trial. J THORAC CARDIOVASC SURG 1992;103:194-9.