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Effect of oxygenated crystalloid cardioplegia on the functional and metabolic recovery of the isolated perfused rat heart
J
THORAC CARDIOVASC SURG
91:259-269, 1986
Effect of oxygenated crystalloid cardioplegia on the functional and metabolic recovery of the isolated perfused rat heart The IR of an oxygenated crystalloid cardioplegic solurion to improve myocardial preservation during elective cardiac arrest was evaluated with the isolated perfused rat heart used as a model. Experiments were conducted at 4° C and 20° C. The oxygen te~ion of the nonoxygenated and oxygenated cardioplegic solutio~ averaged 117 and 440 mm Hg, respectively. At 4° C, the adenosine triphosphate content of hearts subjected to 120 minutes of oxygenated cardioplegia was significantly higher than that of the nonoxygenated cardioplegia group. However, functional recovery during reperfusion was similar for both groups. At 20° C, the myocardial adenosine triphosphate concentration decreased at a significantly faster rate during ischemia in the group receiving nonoxygenated cardioplegia compared with the oxygenated cardioplegia group. Hearts subjected to 180 minutes of ischemia with oxygenated cardioplegia had a normal ultrastructural appearance whereas hearts subjected to 120 minutes of nonoxygenated cardioplegia showed severe ischemic damage. Myocardial functional recovery in the group receiving oxygenated cardioplegia exceeded that of the group receiving nonoxygenated cardioplegia. The use of myocardial adenosine triphosphate concentration at the end of the ischemic period to predict subsequent cardiac output, peak systolic pressure, and total myocardial work showed significant positive correlatio~.
A. Coetzee, F.F.A.R.C.S., F.FA (SA), Ph.D., J. Kotze, B.Sc.Honns., J. Louw, and A. Lochner, D.Sc., Ph.D., Tygerberg, South Africa
h e recovery of the myocardium after ischemic arrest has been demonstrated to be directly related to the preservationof the adenosine triphosphate (ATP) molecules.' In clinical practice, various methods are employed to prevent reduction of myocardial ATP and creatine phosphate (CP) during cardiopulmonary bypass. Hypothermia confers protection by reducing the metabolic rate of the heart, and potassium-containing cardioplegic solutions are used to achieve and maintain arrest in diastole. 2-4 It would also appear that the combination of hypothermia and potassium-induced From the Department of Anesthesiology and MRC Unit for Molecular and Cellular Cardiology, and MN R Unit for Electron Microscopy, University of Stellenbosch Medical School, Tygerberg, South Africa. Supported by the Medical Research Council of South Africa. Received for publication Nov. 20, 1984. Accepted for publication March 27, 1985. Addressfor reprints: Prof. A. Coetzee, Department ofAnesthesiology, P.O. Box 63, Tygerberg, 7505, South Africa.
arrest gives better protection to the ischemic myocardium than each of these perturbations alone.>" Furthermore, multidose cardioplegia has been shown to enhance ATP and CP preservation during ischemic arrest.':'? To supply oxygen to the myocardium during a period of ischemic arrest, blood cardioplegia has been advocated and good results were reported.":" However, because of the leftward shift of the oxyhemoglobin dissociation curve" and the increase in viscosity during hypothermia, alternative methods for myocardial oxygenation are still being sought. In view of these disadvantages of blood cardioplegia and the theoretical advantages of the use of oxygenated crystalloid solutions, we evaluated the effect of intermittent administration of the latter (at 4° C and 20° C) on the metabolic state and ultrastructural appearance of the isolated perfused rat heart subjected to different periods of ischemia. Furthermore, the capacity of such hearts to recover during reperfusion at 37° C was determined by the following parameters: tissue high-energy phosphate contents, ultrastructure, and functional recovery. 259
The Journal of Thoracic and Cardiovascular Surgery
2 6 0 Coetzee et al.
Methods One hundred fifty-two male Wistar rats, weighing 300 to 430 gm, were allowed free access to food and water before they were anesthetized with sodium pentobarbital (Nembutal) (25 to 30 mg per rat). The hearts were removed and underwent retrograde perfusion with the modified Langendorff technique (nonworking hearts) for 10 minutes at 37° C. The perfusate consisted of the following (in mmoljL): NaCl, 119; NaHC0 3, 24.9; KCl, 4.74; KHz P04, 1.19; MgS04; 0.6; Na z S04' 0.59; CeCl; 2.5; glucose 10. After 10 minutes, the preparation was converted into a working heart model according to the method of Neely and associates" as modified by Opie, Mansford, and Owen." This entailed cannulation of the left atrium and allowed for a left atrial filling pressure of 12 em HzO. The left ventricle was then allowed to eject against a 100 em water column. After 10 minutes, the working preparation was characterized in terms of aortic flow, cardiac output, heart rate, kinetic work, pressure work, total work, and peak systolic pressure. IS Results obtained during this period will subsequently be referred to as controls. Aortic flow and coronary blood flow were measured manually and aortic pressure was measured with a Statham P23 D6 pressure transducer. Measurements were made on a research recorder (Model DR-8, Electronics for Medicine, Honeywell Inc.). After the 20 minute control period, hearts were switched to a system that allowed for administration of a cold cardioplegic solution (Plasma-Lyte B plus 25 mmoljl K+: Na+ 130 mmoljL, K+ 30 mmol/L, Mg++ 3 mmoljL, Cl 109 mmoljL, HCO- 28 mmoljL, pH 7.4, osmolality 273 mOsm), which was given as 15 ml/kg body weight. Accurate temperature control was achieved by surrounding the heart with a double-walled glass chamber kept at the desired temperature by a Lauda K4R Electronic circulating water bath. Cardioplegic solution was delivered at a pressure of 40 mm Hg and a rate of 3 to 4 nil/min. Thereafter, the arrested hearts were intermittently reperfused for 1 minute at 10 minute intervals. At the end of the hypothermic ischemic period, the hearts were reperfused in the Langendorff nonworking mode with Krebs-Hense1eit buffer (at 37° C) for 10 minutes before switching to the working heart model. Measurements of coronary and aortic flow rates as well as recordings of mechanical activity were made after 20 minutes of reperfusion. The hearts were subsequently freeze-clamped with Wollenberger tongs and stored in liquid nitrogen until such time as high-energy phosphate analyses could be effected, as described by Edoute and colleagues." Myocardial ATP
and CP contents were expressed in micromoles times gram of dry weight:". Ultrastructural studies were done on two or three hearts from each group studied at 20° C. Hearts were perfusion-fixedand the tissue processedwas as previously described from this laboratory." Four specimens (two subepicardial and two subendocardial) from each heart were examined and six randomly selected micrographs were analyzed for each specimen. Only qualitative analyses of the hearts will be represented in this article. Experimental design All sections described consist of two subgroups: one group received oxygenated cardioplegia (oxygen tension = 440 ± 27.8 mm Hg) whereas the other group received nonoxygenated cardioplegia (oxygen tension = 117.0 ± 13.2 mm Hg). These values represent the mean and standard error (SE) for both the 4° C and 20° C groups. Group I, control. To obtain baseline values for myocardial ATP and CP contents, hearts were perfused in a retrograde fashion for 10 minutes followed by a 10 minute period as a working heart (at 37° C). Thereafter, the hearts were freeze-clamped and analyzed. Group II, (4° C, no reperfusion) (Table I). After a period of perfusion similar to Group I, hearts were arrested with the cardioplegic solution (cooled to 4°C) and were kept for periods of 30, 60, and 120 minutes at 4° C while cardioplegic solution was infused intermittently. After the ischemic period, hearts were freezeclamped and stored for analysis. Group III, 4° C, with reperfusion (Table II). This group is similar to Group II except that at the end of the ischemic period the hearts were reperfused in a retrograde fashion for a period of 10 minutes followed by perfusion in the working mode for 10 minutes (at 37° C). During the latter period myocardial function was quantified and the hearts were subsequently freezeclamped and analyzed for ATP and CPo Because little difference in function could be demonstrated between the oxygenated and nonoxygenated groups after 120 minutes of ischemia, it was decided to extend the period of ischemia for the remainder of the experiments to 150 and 180 minutes. Group IV, 20° C, no reperfusion (Table III). The protocol of this group is similar to that of Group II except that the cardioplegic solution as well as the hearts during the ischemic period were kept at 20° C. In this series the effects of 90, 120, 150, and 180 minutes of ischemia were evaluated.
Volume 91
Oxygenated crystalloid cardioplegia
Number 2 February, 1986
261
Table I. Adenosine triphosphate and creatine phosphate contents of rat hearts after periods of ischemia at 4° C (no reperfusion) Cardioplegia 60 min
30 min
ATP (Ilmol/gm dw)
CP (urnol/gm dw)
Control (N=5)
+02 (N=6)
20,35 ± 1.66 28,23 ±2.71
20.47 ± 1.63 28.49 ±2.84
I
-02 (N=5)
+02 (N=5)
18.81 ±3,30 17.90 ±4.27
14.98*
± 1.14 19.40* ±2.78
I
120 min
-02 (N=5)
+02 (N=6)
14.21* ±0.81 20.96 ±3.76
19.98ttt ± 1.18 25.29ttt ±2.19
-02 (N=6)
I
7.18*** ±1.42 9.34*** ± 1.71
Legend: dw, Dry weight. Asterisks indicate differences between value indicated and control (preischemic) values. Daggers indicate differences between oxygenated (+0,) and nonoxygenated (-0,) cardioplegia groups. *tp < 0.05. **ttp < 0.01. ***tttp < 0.001.
Table II. Adenosine triphosphate and creatine phosphate contents of rat hearts after ischemic arrest at 4 ° C followed by 20 minute reperfusion at 37° C (retrograde: 10 minutes; working model: 10 minutes) Cardioplegia 90 min
ATP (Ilmo1/gm dw)
CP (umcl/gm dw)
120 min
-0
-0
Control (N= 5)
+02 (N=8)
I (N= 27)
+02 (N=6)
2 I (N=6)
20.35 ± 1.66 28.23 ±2.71
16.35 ± 1.23 26.52 ±5.74
15.38* ± 1.37 20.61 ±3.14
17.84 ± 1.93 24,31 ±4.41
16.26 ±0.96 26.01 ±7.83
150 min
+02 (N= 7) 17.06 ± 1.11 26.82 ±2.13
I
180 min
-02 (N= 7)
+02 (N=9)
13.17* ±2.20 22.03 ±2.63
16.19*t ±0.81 23.26 ±2.78
I
-02 (N=9) 12.51** ± 1.01 23.04 ±2.03
Legend: Asterisks indicate difference between value indicated and preischemic (control) values. Daggers indicate difference between group that received oxygenated cardioplegia (+0,) and groups receiving nonoxygenated cardioplegia (-0,). *tp < 0.05. **p < 0.01.
Group V, 20° C, with reperfusion (Table IV). This group was similar to Group III with the exception that the cardioplegic solution and the temperature during the ischemic period was kept at a constant 20° C. Certain stages were omitted in this group. During the 90 minute ischemic period only nonoxygenated cardioplegia was evaluated, because at the 120 minute period of ischemia the nonygenated cardioplegia group showed very little functional recovery (often none); it was not required to repeat this part of the experiment at 180 minutes. Similarly, in the 180 minute experiment, oxygenated cardioplegia only was evaluated, because at 120 minutes of ischemia the nonoxygenated cardioplegia group showed very little functional recovery (often none); it was not required to repeat this part of the experiment at 180 minutes. When results are presented with regard to myocardial function, each heart served as its own control (i.e., the
prearrest function was compared with the postarrest function). Statistical analysis Comparison of data obtained before and after the ischemic periods were done with the paired Student's t test. For comparisons between groups, the unpaired t test was used. A level of p < 0.05 was accepted as significant. All the results are expressed as mean ±
SE. Results Cardioplegia at 4° C (Groups II and III). Myocardial ATP content was significantly reduced (p < 0.05) after 60 minutes of ischemic arrest with both oxygenated and nonoxygenated cardioplegic solutions. Only after a 120 minute ischemic arrest could a difference in the ATP concentrations be demonstrated between the oxygenated and nonoxygenated cardioplegia groups
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Coetzee et al.
Thoracic and Cardiovascular Surgery
Table m. Adenosine triphosphate and creatine phosphate in rat hearts after ischemic arrest at 20° C (no reperfusion) Cardioplegia 90 min (N=5)
(N=6)
I (N=6) -0
20.35 ± 1.66 28.23 ±2.71
22.69t ±1.43 40.78t ±6.88
16.88 ± 1.31 21.05 ±3.55
Control ATP (umol/grn dw) CP (umolj'gm dw)
+02
150 min
120 min 2
I
+02
(N= 6)
20. I2ttt ±0.86 25.42t ±5.10
-02
I
+02
(N= 6)
(N=6)
8.38*** ±1.39 11.59** ±2.27
19.86ttt
± 1.74 33.84ttt ±4.17
180 min
-02
+02
(N=6)
(N= 6)
2.49*** ±0.47 3.61*** ± 1.35
16.90ttt ±3.35 31.55tt ±8.35
I
-02
(N= 6)
0.75** ±0.18 0.99 ±0.22
Legend: Asterisks indicate differences obtained between control (preischemic) hearts and values indicated. Daggers indicate differences between group receiving oxygenated cardioplegia (+0,) and group receiving nonoxygenated cardioplegia (-0,). Values are mean ± standard error.
*t p < 0.05. **tt p < 0.01 ***ttt p < 0.001.
Table IV. Adenosine triphosphate and creatine phosphate contents in rat hearts after ischemic arrest at 20° C and 10 minute passive reperfusion at 37° C and 10 minute working heart model
-
Cardioplegia 90 min
ATP (umol/gm dw) CP (umol/gm dw)
120 min
Control
-02
+02
(N=5)
(N=6)
(N=6)
20.35 ± 1.66 28.23 ±2.71
14.64 ± 1.83 29.11 ±3.31
20.67tt ± 1.51 35.33 ±4.57
I
150 min
-02
+02
(N=6)
(N=6)
11.81** ± 1.89 27.95 ±4.24
20.13ttt ±0.68 26.81 ±3.26
I
180 min
-02
+02
(N=6)
(N=6
7.19*** ±1.49 19.69 ±4.14
20.86 ± 1.13 23.67 ± 1.57
Legend: Asterisks indicate differences between values indicated and preischemic (control) values. Daggers indicate differences between hearts receiving oxygenated cardioplegia (+0,) and hearts receiving nonoxygenated cardioplegia (-0,). Values are mean ± standard error.
**tt p < 0.01. ***ttt p < 0.001.
(Table I). After reperfusion (Group III) the ATP content of hearts receiving oxygenated cardioplegia was significantly higher than that of hearts receiving nonoxygenated cardioplegia after 180 minutes of ischemia (Table 11). After 120 minutes of ischemia, the CP levels of hearts receiving oxygenated cardioplegia were significantly higher than those of hearts receiving nonoxygenated cardioplegia. Once reperfusion was effected, the CP levels returned to control values at all time intervals studied. In all hearts studied, mechanical function during reperfusion was significantly lower than the corresponding preischemic control values. However, differences between the oxygenated and the nonoxygenated cardioplegic groups could only be demonstrated for aortic output and heart rate at 120 minutes and peak systolic pressure at 180 minutes (Table V). The presence of oxygen in the cardioplegic solution had no effect on the total work performed during reperfusion.
Cardioplegia at 20° C (Groups IV and V). Significant differences could be demonstrated for the ATP content of the nonoxygenated and the oxygenated cardioplegia groups for each period of ischemic arrest examined in this part of the protocol. In the oxygenated group the ATP content was better preserved compared with that of the nonoxygenated group (Fig. 1, Table III). Of particular significance is the observation that the high-energy phosphate levels of hearts subjected to 180 minutes of ischemic arrest with oxygenated cardioplegia were similar to those of control hearts (Table III). After reperfusion the differences in ATP levels between the oxygenated and nonoxygenated cardioplegia groups were still present (Table IV). A significant negative correlation was obtained when the decrease in the ATP concentration was compared with ischemic arrest time (Fig. 2). The regression analysis for the oxygenated cardioplegia group was as follows: ATP = -0.OO6( ± 0.002) . minutes + 3.597 (F = 8.622, df = 1,2). For the nonoxygenated cardiople-
Volume 91 Number 2
Oxygenated crystalloid cardioplegia
February, 1986
26 3
20'C 15 MIN REPERFUSION AT 3l'C
,..tt-,
25
,ttt-,
20 ATP rnicromot.q"" (dry weight)
15 10
5
CONTROL
ISCHEMIC ARREST (minutes)
Fig. 1. Myocardial adenosine triphosphate (ATP) concentration after ischemic arrest at 20° C and 20 minutes of reperfusion at 37° C. Asterisks indicate differences between value indicated and preischemic (control) values whereas daggers indicate differences between groups receiving oxygenated (+02) and nonoxygenated (-0 2) cardioplegic solutions. tt **p < 0.01. ttt ***p < 0.001. Values are mean ± standard error.
20'C CARDIOPLEGIA
25 20
I3::---1 - - 1 _ _1 ...J
15 ATP micrornol.q'" (dry weight)
10
°z ° 0: I-
--I
I ***
I
(J
5 0
I
I
90
120
•
+°2
r=-o.92
N = 23
P
. -0 2
r=-o.93
N = 24
p
***-, x***
~
150
180
ISCHEMIC ARREST (minutes)
Fig. 2. Decline of myocardial ATP levels after various ischemic arrest periods at 20° C. The asterisks indicate differences for particular values indicated and control (preischemic) values. The oxygenated cardioplegia group could be characterized as: y = -0.006 (±0.002)x + 3.597 (F = 8.622, df = 1,2). The nonoxygenated group could be expressed as y = -0.021 (±0.004)x + 3.746 (F = 22.16, df = 1,2). The slopes of the two regression analyses performed differed significantly at the 5% level (F = 9.504, df = 1,4).
gia group the analysis showed the following: ATP = -0.021 (±0.004). minutes + 3.746 (F = 22.16, df= 1,2). The slopes of the two regression analyses differed at the 5% level (F = 9.504, df = 1,4). Myocardial function during reperfusion was maintained significantly better in the group receiving the oxygenated cardioplegic solution. After 120 minutes of ischemia, hearts receiving the nonoxygenated cardioplegic solution were mostly nonfunctional (Table VI). After 150 minutes none of these hearts was able to produce aortic flow. When the ATP concentration at the end of the ischemic period was used to predict postischemic func-
tion, significant correlations could be demonstrated for cardiac output (CO) (Fig. 3), peak systolic pressure (PSP) (Fig. 4), and total work (Wt). ATP/CO (r = 0.87, P < 0.001, N = 36): CO = 2.70( ±0.53) . ATP - 5.39 F = 26.36, df = 1,4 ATP/PSP (r = 0.99, P < 0.001, N = 36): PSP = 6.06( ± 0.33 . ATP - 14.82 F = 58.69, df = 1,5 ATP/Wt (r = 0.87, P < 0.001, N = 36): Wt = 0.724(±O.l4) . ATP - 3.23 F = 25.87, df = 1,4
The Journal of Thoracic and Cardiovascular
2 6 4 Coetzee et al.
Surgery
20' CARDIOPLEGIA 60
50 40 CARDIAC OUTPUT ml.mln'" 30 20 r= 0.87
10
N = 36
P <0,001
o
10
5
15
20
25
ATP mlcromol.g- 1 (dry weighl)
Fig. 3. ATP concentrations at the end of the various 20° C ischemic periods (i.e., no reperfusion) were plotted against the cardiac output attained after a 20 minute reperfusion period at 37° C. The linear regression could be expressed as: y = 2.70( ±0.53)x - 5.39 (F = 26.36, df = 1,4). 20' CARDIOPLEGIA
120 100 80 PEAK SYSTOLIC PRESSURE mmHg
60 40 20
0
/
5
r=0.99 p<0.001
10
15
20
N=36
25
ATP mlcromol.g-1 (drywelghl)
Fig. 4. ATP concentrations in the myocardium after the various 20° C ischemic arrest periods are plotted against the peak systolic pressure developed after 20 minutes of reperfusion at 37° C. The linear regression was: y = 6.06 (±0.33)x - 14.82 (F = 337.3, df = 1,5).
Electron microscopic findings. Figs. 5 and 6 are representative examples of the ultrastructure of the subepicardial and subendocardial layers of a heart subjected to 120 minutes of ischemia and nonoxygenated cardioplegia. The subepicardial layer showed considerable intramyofibrillar and intermyofibrillar edema. Mitochondria appeared rounded but otherwise normal. Sarcomeres were contracted and glycogen was absent. However, the subendocardial layer showed marked ischemic damage with severe mitochondrial swelling, clearing of the matrix, and rupture of the cristae. Rupture of mitochondrial outer membrane was seen. Sarcomeres were contracted and glycogen was absent. Hearts subjected to 180 minutes of ischemic arrest with oxygenated cardioplegia exhibited normal ultra-
structure of both layers. Fig. 7 shows a typical example of the ultrastructure of the subendocardium of such a heart. Discussion
The rationale for the use of oxygenated cardioplegic solutions is based on the need to supply oxygen to the myocardium during elective ischemic arrest periods while the subject is undergoing cardiopulmonary bypass. Use of blood cardioplegia to achieve this purpose appears to have certain disadvantages. Cold blood has a P so' that is detrimental to tissue oxygen delivery (calcu*Oxygen tension at which hemoglobin is half saturated with oxygen.
Volume 91 Number 2 February. 1986
Fig. 5. Subepicardium of a heart subjected to 120 minutes of nonoxygenated cardioplegia at 20° C. Intramyofibrillar and intermyofibrillar edema and contracted sarcomeres are present. Glycogen is absent. (Magnification X4,900.)
lated P so at 20° C = 18 mm Hg, 10° C = 15 mm Hg, and 5° C = 13.5 mm Hg I5); and since blood viscosity has an inverse relationship with temperature," oxygen delivery at a cellular level may be jeopardized. The physical laws governing oxygen flux in crystalloids predict that (1) oxygen solubility increases with a decrease in temperature, (2) all the oxygen in the solution is available for oxygen utilization, and (3) the viscosity of a crystalloid solution is affected to a lesser extent. The differences in oxygen availability from blood compared with an oxygenated crystalloid solution is evident from the following data: At 10° C an oxygenated crystalloid solution released 4.03 ± 0.07 ml oxygen/loo ml solution, which was significantly higher than the oxygen released from blood at the same temperature (hemoglobin 5.3 gm/dl, oxygen content 9.76 ± 0.48 ml oxygen/loo ml blood; oxygen released = 3.6 ± 0.1 ml/loo ml)." Therefore, blood cardioplegia cannot be accepted as the fmal answer to the issue of oxygen supply during hypothermic arrest. The feasibility of the use of an oxygenated crystalloid solution for cardioplegia was evaluated by Bodenhamer and colleagues" in 1983. In a canine heart model, these authors demonstrated a significantly improved preserva-
Oxygenated crystalloid cardioplegia
265
Fig. 6. Subendocardium of a heart subjected to 120 minutes of nonoxygenated cardioplegia at 20° C. Marked ischemic damage with severe mitochondrial swelling, clearing of the matrix, and the rupture of the cristae and outer membrane are visible. Sarcomeres are contracted and glycogen is absent. (Magnification X4,900.)
tion of myocardial function and ATP concentration after the use of an oxygenated crystalloid cardioplegic solution. Their biochemical fmdings were supported by electron microscopic data. The results obtained in our study showed that oxygenation of the cardioplegic solution at 4° C resulted in better preservation of tissue high-energy phosphates (Tables I and II). However, the differences were not reflected in subsequent myocardial function, for only at 180 minutes of ischemia were peak systolic pressures attained significantly higher in the group receiving oxygenated cardioplegia (Table V). When ATP concentrations at the end of the ischemic arrest period were correlated with cardiac output, peak systolic pressure and total work, no significant correlation could be obtained. The failure of 4°C oxygenated cardioplegia to improve functional recovery during reperfusion is probably because temperatures below 15° C are detrimental to rat hearts. Cat and rat hearts showed improved mechanical recovery when hearts were maintained at 15° C (compared with 4° C).24,25 This result is substan-
2 6 6 Coetzee et al.
Fig. 7. Subendocardium of a heart subjected to 180 minutes of oxygenated cardioplegia at 20° C. Normal ultrastructure. (Magnification X4,900.)
tiated by our finding that functional recovery of hearts subjected to 20° C oxygenated cardioplegia was significantly better than that of hearts subjected to a similar period of ischemia and 4 ° C cardioplegia (compare Tables V and VI). The lowest value for myocardial ATP content obtained at the end of an ischemic period (120 minutes of cardioplegia without additional oxygen) was 35% of the control (Group I) value. However, myocardial function during reperfusion was well preserved. These results differ slightly from those of Reibel and Revette," who reported that rat hearts with an ATP concentration less than 35% of control were unable to produce aortic output during reperfusion. Our results are clearly also at variance with the concept that reduction of ATP below 50% to 60% of control ATP is not compatible with survival of the myocardium. 3 Results obtained from our study conducted at 20° C are probably more applicable to the clinical situation. Patients at our institution who are on cardiopulmonary bypass are usually maintained at 26° C to 28° C, cardioplegic solution (at 4° C) is given every 20 minutes at a maximum, and the temperature of the operating room averages 20° C to 21° C. These circumstances suggest that myocardial temperature will be closer to 20° C than 4°C; this is especially true for the
The Journal of Thoracic and Cardiovascular Surgery
hypertrophied myocardium. In contrast to the results obtained at 4 ° C, the slope of the regression analysis for the group receiving nonoxygenated cardioplegic solution at 20° C was much steeper than for the group receiving cardioplegic solution plus additional oxygen (Fig. 2). At 180 minutes, the myocardial ATP content for the latter group did not differ significantly from the control value (Table III). The beneficial effects of the oxygenated crystalloid cardioplegic solution were also evident from the excellent preservation of myocardial microanatomy after exposure of the heart to 180 minutes of ischemia (Fig. 7). Also in this section of the protocol (20° C), it was demonstrated that a significant correlation existed between the developed peak systolic pressure, cardiac output, and total work, during reperfusion and the post-ischemic arrest ATP concentration (Figs. 3 and 4). It is therefore obviousthat the functional recoveryof the rat heart at 20° C is related to the preservation of the ATP molecules, and this observation adds weight to the argument that every possible step should be taken to allow for the maximum preservation of ATP molecules during ischemic arrest. After 90 minutes of ischemia at 20° C, hearts perfused with cardioplegic solution without additional oxygen developed acceptable peak systolic pressure, but cardiac output was reduced to approximately 50% of control values. In the period after cardiopulmonary bypass an inadequate cardiac output will lead to venous desaturation and an increase in blood lactate, a decrease in pH, and eventually circulatory decompensation. The peak systolic pressure developed after 90 minutes of ischemia may give a false impression of the cardiovascular dynamics, and we therefore suggest that oxygenation of the myocardium during a 90 minute ischemic period is as important as for 180 minute ischemic arrest periods. Our previous work showed that work performance during reperfusion was closely associated with both ultrastructure and tissue high-energy phosphate concentration." The ATP conservation that resulted from the use of oxygenated cardioplegia is reflected in the excellent myocardial functional and ultrastructural recovery after a 3 hour period of ischemia in the rat heart (Table VI, Fig. 7). Similarly, work done on canine hearts demonstrated superior recovery after 4 hours of ischemia if a crystalloid cardioplegic solution with oxygen was used." A study conducted on human beings during cardiopulmonary bypass," which used blood cardioplegia at 15° C for a mean period of 77 minutes of ischemia, demonstrated a 15% increase in ATP concentration at the end of ischemia when compared with control values.
Volume 91
Oxygenated crystalloid cardioplegia
Number 2
267
February, 1986
Table V. Mechanical performance of rat hearts subjected to 4' C ischemic arrest followed by reperfusion at 37" C (retrograde: 10 minutes; working model: 10 minutes) Cardioplegia and reperfusion
Control (N=54) Aortic now (mi. min-I) Heart rate (beats· min-I) Peak systolic pressure (mm
I
+0,
(N= 7)
-02
+02
(N=6)
(N=5)
41.35 ±1.29 298.63 ±6.80 119.81 ±2.68
10.50** ±6.61 253.99 ±51.92 81.11 ± 17.57
24.17** ±6.21 273.70 ± 15.79 108.31 ±6.17
9.22***t ±2.04 235.50**t ± 21.70 107.60** ±4.67
15.24 ±0.55 0.51 ±0.06 15.79 ±0.51
7.58** ±2.64 0.18 ±0.15 7.76** ±2.78
9.89** ±2.05 0.15 ±0.06 10.04** ±2.09
6.80*** ± 1.06 0.15** ±0.06 6.96*** ± 1.11
I
ISO min
150 min
120 min
90 min
-02
-02
I
+02
+02
I
-02
(N=6)
(N= 11)
25.15** ±4.49 295.42 ± 29.59 94.70** ±5.65
14.88** ±4.08 240.33 ± 21.57 97.70** ±5.33
11.57*** ±3.32 264.09** ± 16.72 97.43t ±2.03
6.17*** ±2.26 279.18 ± 13.37 83.33*** ±6.35
9.44** ± 1.65 0.19** ±0.05 7.64** ±0.64
6.82** ± 1.24 0.10 ±0.03 6.92** ± 1.27
6.12*** ±0.93 0.08** ±0.03 6.20*** ±0.94
4.08*** ±0.84 0.03** ±0.01 4.10*** ±0.86
(N=5)
(N=5)
23.25** ±3.75 315.73 ± 11.46 109.95 ±3.08 10.89 ± 1.28 0.55 ±0.17 9.54** ±1.45
(N=9)
Hg) Pressure work (mW) Kinetic work (mW) Total work (mW)
l.egend: Asterisks indicate difference between values indicated and control (prcischernic) values. Daggers indicate differences between groups receiving oxygenated (+0 2 ) and nonoxygenatcd (-0 2) cardioplegia. Values are mean ± standard error.
°tp < 0.05.
"p < 0.01.
"op < 0.001.
Table VI. Mechanical performance of rat hearts after ischemic arrest at 20' C and passive reperfusion at 37" C for 10 minutes and as a working heart model for 10 minutes Cardioplegia 90 min Control (N=6)
Aortic now (mi. min-I) Heart rate (beats. min-I) Peak systolic
pressure
43.58 ± 1.39 281.00 ±2.68 109.73 ±2.98
120 min
-02
+02
(N=6)
(N=6)
11.8** ±3.42 306.58 ± 18.55 93.57** ±4.13
37.83ttt ±5.34 292.82t ±5.41 99.65*t ±3.15
I
150 min
-02
+02
(N=6)
(N=6)
1.16*** ± 1.17 107.15* ±48.18 33.80** ± 18.38 1.23*** ±0.81 om** ±0.01 1.24*** ±0.82
ISO min
-0,
+02
(N=6)
(N=6
32.90*ttt ±5.12 285.08ttt ± 10.17 106.20*ttt ±2.49
0*** 0 0*** 0 0*** 0
30.17** ±3.61 275.63 ± 15.20 90.93*** ± 15.21
12.25*ttt ± 1.14 0.14*tt ±0.04 13.81tt ±2.59
0*** 0 0** 0 0*** 0
9.55** ±0.87 0.13 ±0.05 9.63** ±0.92
I
(mm Hg) Pressure work (mW) Kinetic work (mW) Total work (mW)
14.82 ±0.86 0.21 ±0.02 15.04 ±0.88
6.03*** ±0.97 0.06** ±0.03 6.11*** ±0.98
12.00ttt ± 1.57 0.13*t ±0.03 11.05*ttt ± 1.71
Legend: Asterisks indicate differences between values indicated and (preischemic) controls. Daggers indicate differences between group receiving oxygenated cardioplegia (+0,) and hearts receiving nonoxygenated cardioplegia (-0,). Values are mean ± standard error.
otp < 0.05 . ..ttp
< 0.01.
..otttp < 0.00 I.
The 90 minute ischemic arrest period from our study at 20° C showed a 12% increase in ATP at the end of the ischemic arrest period. Although not totally comparable in terms of animals used, time perfused, and temperature used, the use of an oxygenated crystalloid cardio-
plegic solution in rats compares favorably to that of blood used in human beings. ATP levels of hearts reperfused after 90, 120,and 150 minutes of ischemia while receiving nonoxygenated 20° C cardioplegic solution remained low whereas CP
2 6 8 Coetzee et al.
content increased substantially. Similar results have been reported before in hearts reperfused after normothermic ischemic cardiac arrest. 20,27.28 The importance of oxygen supply has been highlighted in this study and confirmed the results obtained by Bodenhamer and colleagues." We suggest that this method could be used safely in patients; until the perfluorocarbon solutions are generally available," oxygenated crystalloid cardioplegia could be used to improve the ATP concentration of the myocardium during ischemic arrest. The next step in the examination of this model is the evaluation of a graded increase in the oxygen content in the crystalloid solution as well as the evaluation of the effect of additional substrate in the solution. Theoretically, the preservation of myocardial ATP, and hence function, should increase; this may extend the time for ischemic arrest during cardiopulmonary bypass even further than what has been accepted as a maximum to date. A. Bunn, Ph.D., of the Department of Medicine of the University of Stellenbosch provided assistance with the statistical analysis. REFERENCES
2
3
4
5
6
7
Hearse DJ, Stewart DA, Chain EB: Recovery from cardiac bypass and elective cardiac arrest. Circ Res 35:448-457, 1974 Roberts AJ, Abel RM, Alonso DR, Subramanian VA, Paul JS, Gay W A: Advantages of hypothermic potassium cardioplegia and superiority of continuous versus intermittent aortic cross-clamping. J THORAC CARDIOVASC SURG 79:44-58, 1980 Bretschneider HJ, Hubner G, Knoll D, Lohr B, Nordbeck H, Spieckerman PG: Myocardial resistance and tolerance to ischemia. Physiological and biochemical basis. J Cardiavase Surg 16:241-260, 1975 Hearse DJ, Stewart DA, Braimbridge MV: Hypothermic arrest and potassium arrest. Metabolic and myocardial protection during elective cardiac arrest. Circ Res 36:481489, 1975 Hearse DJ, Stewart DA, Braimbridge MV: The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the rat. J THORAC CARDIOVASC SURG 79:39-43, 1980 Rosenfeldt FL, Hearse DJ, Cankovic-Darracott S, Braimbridge MV: The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the dog. J THORAC CARDIOVASC SURG 79:29-38, 1980 Lucas SK, Elmer EB, Flaherty JT, Prodromos CC, Buckley BH, Gott VL, Gardner TJ: Effect of multidose potassium cardioplegia on myocardial ischemia, return of ventricular function, and ultrastructural preservation. J THORAC CARDIOVASC SURG 80: 102-11 0, 1980
The Journal of Thoracic and Cardiovascular Surgery
8 Engelman RM, Anvil J, O'Donoghue MJ, Levitsky S: The significance of multidose cardioplegia and hypothermia in myocardial preservation during ischemic arrest. J THORAC CARDIOVASC SURG 75:555-563, 1978 9 Nelson RL, Fey KH, Follette DM, Livesay JJ, De Land EC, Maloney,lV, Buckberg GD: The critical importance of intermittent infusion of cardioplegic solution during aortic cross-clamping. Surg Forum 27:241-243, 1976 IO Flaherty JT, Jacobus WE, Weisfeldt ML, Hollis DL, Gardner TJ, Gott VL: Use of 31 phosphorus nuclear magnetic resonance to assess myocardial protection during global ischemia (Abstr). Am J Cardiol 43:362, 1979 II Follette DM, Mulder DG, Maloney JV, Buckberg GD: Advantage of blood cardioplegia over continuous coronary perfusion or intermittent ischemia. Experimental and clinical study. J THORAC CARDIOVASC SURG 76:604-619, 1978 12 Cunningham IN, Adams PX, Knopp EA, Bauman FG, Snively SL, Gross RI, Nathan 1M, Spencer FC: Preservation of ATP, ultrastructure, and ventricular function after aortic cross-clamping and reperfusion. J THORAC CARDlOVASC SURG 98:708-720, 1979 13 Singh A, Farrugia R, Teplitz C, Karlson KE: Electrolyte versus blood cardioplegia. Randomized clinical and myocardial ultrastructural study. Ann Thorac Surg 33:218227, 1982 14 Catinella FP, Cunningham IN, Knopp EA, Laschinger JC, Spencer FC: Preservation of myocardial ATP. Comparison of blood vs crystalloid cardioplegia. Chest 83:65065, 1983 15 Severinghaus JW: Oxyhemoglobin dissociation curve correction for temperature and pH variation in human blood. J Appl Physiol 13:485-486, 1958 16 Neely JR, Liebermeister H, Battersby EJ, Morgan HE: Effects of pressure development on oxygen consumption by isolated rat hearts. Am J Physiol 212:804-814, 1967 17 Opie LH, Mansford KRL, Owen P: Effect of increased heart work on glycolysis and adenine nucleotide in the perfused heart of normal and diabetic rats. Biochem J 124:475-490, 1971 18 Kannengieser G J, Opie LH, Van der Werff TJ: Impaired cardiac work and oxygen uptake after reperfusion of regionally ischemic myocardium. J Mol Cell Cardiol 11:197-207,1979 19 Edoute Y, Graney D, Sanan D, Kotze JCN, Lochner A: Effects of propranolol on myocardial ultrastructure, mitochondrial function and high energy phosphates of working rat hearts with coronary artery ligation. J Mol Cell Cardiol 13:619-639, 1981 20 Edoute Y, Van der Merwe E, Sanan D, Kotze JCN, Steinman C, Lochner A: Normothermic ischemic cardiac arrest in the isolated work rat heart. Circ Res 53:663-678, 1983 21 Kanter GS: Hypothermic hemoconcentration. Am J Physiol 214:856-859,1968 22 Digerness SB, Vanini V, Wideman FE: In vitro comparison of oxygen availability from asanguineous and sanguin-
Volume 91 Number 2 February, 1986
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24
25
26
eous cardioplegic media. Circulation 64:Suppl 2:80-83, 1981 Bodenhamer RM, DeBoer LW, Giffin G, O'Keefe D, Fallon IT, Hoetz TH, Haas GS, Daggett WM: Enhanced myocardial protection during ischemic arrest. J THORAC CARDIOVASC SURG 85:769-780, 1983 Tyers GFO, Williams EH, Hughes HC, Todd GJ: Effect of perfusate temperature on myocardial protection from ischemia. J THORAC CARDIOVASC SURG 73:766-771, 1977 Flaherty JT, Schaff HV, Goldman RA, Gott VC: Metabolic and functional effects of progressive degrees of hypothermia during global ischemia. Am J Physiol 236:H839-H845, 1979 Reibel DK, Rovetto MJ: Myocardial ATP synthesis and
Oxygenated crystalloid cardioplegia
269
mechanical function following oxygen deficiency. Am J Physiol 234:H620-H624, 1978 27 Swain JL, Sabina RL, McHale PA, Greenfield JC, Holmes EW: Prolonged myocardial nucleotide depletion after brief ischemia in the open-chest dog. Am J Physiol 242:H818-H826, 1982 28 Kloner RA, DeBoer LWV, Darsee JR, Ingnall JS, Hale S, Tumas J, Braunwald E: Prolonged abnormalities of myocardium salvaged by reperfusion. Am J Physiol 242:H818-H826, 1982 29 Flaherty JT, Jaffm JH, Magovern GJ, Kanter KR, Gardner TJ, Miceli MV, Jacobus WE: Maintenance of aerobic metabolism during global ischemia with perfluorocarbon cardioplegia improves myocardial preservation. Circulation 69:585-592, 1984
THORAC CARDIOVASC SURG
91:259-269, 1986
Effect of oxygenated crystalloid cardioplegia on the functional and metabolic recovery of the isolated perfused rat heart The IR of an oxygenated crystalloid cardioplegic solurion to improve myocardial preservation during elective cardiac arrest was evaluated with the isolated perfused rat heart used as a model. Experiments were conducted at 4° C and 20° C. The oxygen te~ion of the nonoxygenated and oxygenated cardioplegic solutio~ averaged 117 and 440 mm Hg, respectively. At 4° C, the adenosine triphosphate content of hearts subjected to 120 minutes of oxygenated cardioplegia was significantly higher than that of the nonoxygenated cardioplegia group. However, functional recovery during reperfusion was similar for both groups. At 20° C, the myocardial adenosine triphosphate concentration decreased at a significantly faster rate during ischemia in the group receiving nonoxygenated cardioplegia compared with the oxygenated cardioplegia group. Hearts subjected to 180 minutes of ischemia with oxygenated cardioplegia had a normal ultrastructural appearance whereas hearts subjected to 120 minutes of nonoxygenated cardioplegia showed severe ischemic damage. Myocardial functional recovery in the group receiving oxygenated cardioplegia exceeded that of the group receiving nonoxygenated cardioplegia. The use of myocardial adenosine triphosphate concentration at the end of the ischemic period to predict subsequent cardiac output, peak systolic pressure, and total myocardial work showed significant positive correlatio~.
A. Coetzee, F.F.A.R.C.S., F.FA (SA), Ph.D., J. Kotze, B.Sc.Honns., J. Louw, and A. Lochner, D.Sc., Ph.D., Tygerberg, South Africa
h e recovery of the myocardium after ischemic arrest has been demonstrated to be directly related to the preservationof the adenosine triphosphate (ATP) molecules.' In clinical practice, various methods are employed to prevent reduction of myocardial ATP and creatine phosphate (CP) during cardiopulmonary bypass. Hypothermia confers protection by reducing the metabolic rate of the heart, and potassium-containing cardioplegic solutions are used to achieve and maintain arrest in diastole. 2-4 It would also appear that the combination of hypothermia and potassium-induced From the Department of Anesthesiology and MRC Unit for Molecular and Cellular Cardiology, and MN R Unit for Electron Microscopy, University of Stellenbosch Medical School, Tygerberg, South Africa. Supported by the Medical Research Council of South Africa. Received for publication Nov. 20, 1984. Accepted for publication March 27, 1985. Addressfor reprints: Prof. A. Coetzee, Department ofAnesthesiology, P.O. Box 63, Tygerberg, 7505, South Africa.
arrest gives better protection to the ischemic myocardium than each of these perturbations alone.>" Furthermore, multidose cardioplegia has been shown to enhance ATP and CP preservation during ischemic arrest.':'? To supply oxygen to the myocardium during a period of ischemic arrest, blood cardioplegia has been advocated and good results were reported.":" However, because of the leftward shift of the oxyhemoglobin dissociation curve" and the increase in viscosity during hypothermia, alternative methods for myocardial oxygenation are still being sought. In view of these disadvantages of blood cardioplegia and the theoretical advantages of the use of oxygenated crystalloid solutions, we evaluated the effect of intermittent administration of the latter (at 4° C and 20° C) on the metabolic state and ultrastructural appearance of the isolated perfused rat heart subjected to different periods of ischemia. Furthermore, the capacity of such hearts to recover during reperfusion at 37° C was determined by the following parameters: tissue high-energy phosphate contents, ultrastructure, and functional recovery. 259
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2 6 0 Coetzee et al.
Methods One hundred fifty-two male Wistar rats, weighing 300 to 430 gm, were allowed free access to food and water before they were anesthetized with sodium pentobarbital (Nembutal) (25 to 30 mg per rat). The hearts were removed and underwent retrograde perfusion with the modified Langendorff technique (nonworking hearts) for 10 minutes at 37° C. The perfusate consisted of the following (in mmoljL): NaCl, 119; NaHC0 3, 24.9; KCl, 4.74; KHz P04, 1.19; MgS04; 0.6; Na z S04' 0.59; CeCl; 2.5; glucose 10. After 10 minutes, the preparation was converted into a working heart model according to the method of Neely and associates" as modified by Opie, Mansford, and Owen." This entailed cannulation of the left atrium and allowed for a left atrial filling pressure of 12 em HzO. The left ventricle was then allowed to eject against a 100 em water column. After 10 minutes, the working preparation was characterized in terms of aortic flow, cardiac output, heart rate, kinetic work, pressure work, total work, and peak systolic pressure. IS Results obtained during this period will subsequently be referred to as controls. Aortic flow and coronary blood flow were measured manually and aortic pressure was measured with a Statham P23 D6 pressure transducer. Measurements were made on a research recorder (Model DR-8, Electronics for Medicine, Honeywell Inc.). After the 20 minute control period, hearts were switched to a system that allowed for administration of a cold cardioplegic solution (Plasma-Lyte B plus 25 mmoljl K+: Na+ 130 mmoljL, K+ 30 mmol/L, Mg++ 3 mmoljL, Cl 109 mmoljL, HCO- 28 mmoljL, pH 7.4, osmolality 273 mOsm), which was given as 15 ml/kg body weight. Accurate temperature control was achieved by surrounding the heart with a double-walled glass chamber kept at the desired temperature by a Lauda K4R Electronic circulating water bath. Cardioplegic solution was delivered at a pressure of 40 mm Hg and a rate of 3 to 4 nil/min. Thereafter, the arrested hearts were intermittently reperfused for 1 minute at 10 minute intervals. At the end of the hypothermic ischemic period, the hearts were reperfused in the Langendorff nonworking mode with Krebs-Hense1eit buffer (at 37° C) for 10 minutes before switching to the working heart model. Measurements of coronary and aortic flow rates as well as recordings of mechanical activity were made after 20 minutes of reperfusion. The hearts were subsequently freeze-clamped with Wollenberger tongs and stored in liquid nitrogen until such time as high-energy phosphate analyses could be effected, as described by Edoute and colleagues." Myocardial ATP
and CP contents were expressed in micromoles times gram of dry weight:". Ultrastructural studies were done on two or three hearts from each group studied at 20° C. Hearts were perfusion-fixedand the tissue processedwas as previously described from this laboratory." Four specimens (two subepicardial and two subendocardial) from each heart were examined and six randomly selected micrographs were analyzed for each specimen. Only qualitative analyses of the hearts will be represented in this article. Experimental design All sections described consist of two subgroups: one group received oxygenated cardioplegia (oxygen tension = 440 ± 27.8 mm Hg) whereas the other group received nonoxygenated cardioplegia (oxygen tension = 117.0 ± 13.2 mm Hg). These values represent the mean and standard error (SE) for both the 4° C and 20° C groups. Group I, control. To obtain baseline values for myocardial ATP and CP contents, hearts were perfused in a retrograde fashion for 10 minutes followed by a 10 minute period as a working heart (at 37° C). Thereafter, the hearts were freeze-clamped and analyzed. Group II, (4° C, no reperfusion) (Table I). After a period of perfusion similar to Group I, hearts were arrested with the cardioplegic solution (cooled to 4°C) and were kept for periods of 30, 60, and 120 minutes at 4° C while cardioplegic solution was infused intermittently. After the ischemic period, hearts were freezeclamped and stored for analysis. Group III, 4° C, with reperfusion (Table II). This group is similar to Group II except that at the end of the ischemic period the hearts were reperfused in a retrograde fashion for a period of 10 minutes followed by perfusion in the working mode for 10 minutes (at 37° C). During the latter period myocardial function was quantified and the hearts were subsequently freezeclamped and analyzed for ATP and CPo Because little difference in function could be demonstrated between the oxygenated and nonoxygenated groups after 120 minutes of ischemia, it was decided to extend the period of ischemia for the remainder of the experiments to 150 and 180 minutes. Group IV, 20° C, no reperfusion (Table III). The protocol of this group is similar to that of Group II except that the cardioplegic solution as well as the hearts during the ischemic period were kept at 20° C. In this series the effects of 90, 120, 150, and 180 minutes of ischemia were evaluated.
Volume 91
Oxygenated crystalloid cardioplegia
Number 2 February, 1986
261
Table I. Adenosine triphosphate and creatine phosphate contents of rat hearts after periods of ischemia at 4° C (no reperfusion) Cardioplegia 60 min
30 min
ATP (Ilmol/gm dw)
CP (urnol/gm dw)
Control (N=5)
+02 (N=6)
20,35 ± 1.66 28,23 ±2.71
20.47 ± 1.63 28.49 ±2.84
I
-02 (N=5)
+02 (N=5)
18.81 ±3,30 17.90 ±4.27
14.98*
± 1.14 19.40* ±2.78
I
120 min
-02 (N=5)
+02 (N=6)
14.21* ±0.81 20.96 ±3.76
19.98ttt ± 1.18 25.29ttt ±2.19
-02 (N=6)
I
7.18*** ±1.42 9.34*** ± 1.71
Legend: dw, Dry weight. Asterisks indicate differences between value indicated and control (preischemic) values. Daggers indicate differences between oxygenated (+0,) and nonoxygenated (-0,) cardioplegia groups. *tp < 0.05. **ttp < 0.01. ***tttp < 0.001.
Table II. Adenosine triphosphate and creatine phosphate contents of rat hearts after ischemic arrest at 4 ° C followed by 20 minute reperfusion at 37° C (retrograde: 10 minutes; working model: 10 minutes) Cardioplegia 90 min
ATP (Ilmo1/gm dw)
CP (umcl/gm dw)
120 min
-0
-0
Control (N= 5)
+02 (N=8)
I (N= 27)
+02 (N=6)
2 I (N=6)
20.35 ± 1.66 28.23 ±2.71
16.35 ± 1.23 26.52 ±5.74
15.38* ± 1.37 20.61 ±3.14
17.84 ± 1.93 24,31 ±4.41
16.26 ±0.96 26.01 ±7.83
150 min
+02 (N= 7) 17.06 ± 1.11 26.82 ±2.13
I
180 min
-02 (N= 7)
+02 (N=9)
13.17* ±2.20 22.03 ±2.63
16.19*t ±0.81 23.26 ±2.78
I
-02 (N=9) 12.51** ± 1.01 23.04 ±2.03
Legend: Asterisks indicate difference between value indicated and preischemic (control) values. Daggers indicate difference between group that received oxygenated cardioplegia (+0,) and groups receiving nonoxygenated cardioplegia (-0,). *tp < 0.05. **p < 0.01.
Group V, 20° C, with reperfusion (Table IV). This group was similar to Group III with the exception that the cardioplegic solution and the temperature during the ischemic period was kept at a constant 20° C. Certain stages were omitted in this group. During the 90 minute ischemic period only nonoxygenated cardioplegia was evaluated, because at the 120 minute period of ischemia the nonygenated cardioplegia group showed very little functional recovery (often none); it was not required to repeat this part of the experiment at 180 minutes. Similarly, in the 180 minute experiment, oxygenated cardioplegia only was evaluated, because at 120 minutes of ischemia the nonoxygenated cardioplegia group showed very little functional recovery (often none); it was not required to repeat this part of the experiment at 180 minutes. When results are presented with regard to myocardial function, each heart served as its own control (i.e., the
prearrest function was compared with the postarrest function). Statistical analysis Comparison of data obtained before and after the ischemic periods were done with the paired Student's t test. For comparisons between groups, the unpaired t test was used. A level of p < 0.05 was accepted as significant. All the results are expressed as mean ±
SE. Results Cardioplegia at 4° C (Groups II and III). Myocardial ATP content was significantly reduced (p < 0.05) after 60 minutes of ischemic arrest with both oxygenated and nonoxygenated cardioplegic solutions. Only after a 120 minute ischemic arrest could a difference in the ATP concentrations be demonstrated between the oxygenated and nonoxygenated cardioplegia groups
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Thoracic and Cardiovascular Surgery
Table m. Adenosine triphosphate and creatine phosphate in rat hearts after ischemic arrest at 20° C (no reperfusion) Cardioplegia 90 min (N=5)
(N=6)
I (N=6) -0
20.35 ± 1.66 28.23 ±2.71
22.69t ±1.43 40.78t ±6.88
16.88 ± 1.31 21.05 ±3.55
Control ATP (umol/grn dw) CP (umolj'gm dw)
+02
150 min
120 min 2
I
+02
(N= 6)
20. I2ttt ±0.86 25.42t ±5.10
-02
I
+02
(N= 6)
(N=6)
8.38*** ±1.39 11.59** ±2.27
19.86ttt
± 1.74 33.84ttt ±4.17
180 min
-02
+02
(N=6)
(N= 6)
2.49*** ±0.47 3.61*** ± 1.35
16.90ttt ±3.35 31.55tt ±8.35
I
-02
(N= 6)
0.75** ±0.18 0.99 ±0.22
Legend: Asterisks indicate differences obtained between control (preischemic) hearts and values indicated. Daggers indicate differences between group receiving oxygenated cardioplegia (+0,) and group receiving nonoxygenated cardioplegia (-0,). Values are mean ± standard error.
*t p < 0.05. **tt p < 0.01 ***ttt p < 0.001.
Table IV. Adenosine triphosphate and creatine phosphate contents in rat hearts after ischemic arrest at 20° C and 10 minute passive reperfusion at 37° C and 10 minute working heart model
-
Cardioplegia 90 min
ATP (umol/gm dw) CP (umol/gm dw)
120 min
Control
-02
+02
(N=5)
(N=6)
(N=6)
20.35 ± 1.66 28.23 ±2.71
14.64 ± 1.83 29.11 ±3.31
20.67tt ± 1.51 35.33 ±4.57
I
150 min
-02
+02
(N=6)
(N=6)
11.81** ± 1.89 27.95 ±4.24
20.13ttt ±0.68 26.81 ±3.26
I
180 min
-02
+02
(N=6)
(N=6
7.19*** ±1.49 19.69 ±4.14
20.86 ± 1.13 23.67 ± 1.57
Legend: Asterisks indicate differences between values indicated and preischemic (control) values. Daggers indicate differences between hearts receiving oxygenated cardioplegia (+0,) and hearts receiving nonoxygenated cardioplegia (-0,). Values are mean ± standard error.
**tt p < 0.01. ***ttt p < 0.001.
(Table I). After reperfusion (Group III) the ATP content of hearts receiving oxygenated cardioplegia was significantly higher than that of hearts receiving nonoxygenated cardioplegia after 180 minutes of ischemia (Table 11). After 120 minutes of ischemia, the CP levels of hearts receiving oxygenated cardioplegia were significantly higher than those of hearts receiving nonoxygenated cardioplegia. Once reperfusion was effected, the CP levels returned to control values at all time intervals studied. In all hearts studied, mechanical function during reperfusion was significantly lower than the corresponding preischemic control values. However, differences between the oxygenated and the nonoxygenated cardioplegic groups could only be demonstrated for aortic output and heart rate at 120 minutes and peak systolic pressure at 180 minutes (Table V). The presence of oxygen in the cardioplegic solution had no effect on the total work performed during reperfusion.
Cardioplegia at 20° C (Groups IV and V). Significant differences could be demonstrated for the ATP content of the nonoxygenated and the oxygenated cardioplegia groups for each period of ischemic arrest examined in this part of the protocol. In the oxygenated group the ATP content was better preserved compared with that of the nonoxygenated group (Fig. 1, Table III). Of particular significance is the observation that the high-energy phosphate levels of hearts subjected to 180 minutes of ischemic arrest with oxygenated cardioplegia were similar to those of control hearts (Table III). After reperfusion the differences in ATP levels between the oxygenated and nonoxygenated cardioplegia groups were still present (Table IV). A significant negative correlation was obtained when the decrease in the ATP concentration was compared with ischemic arrest time (Fig. 2). The regression analysis for the oxygenated cardioplegia group was as follows: ATP = -0.OO6( ± 0.002) . minutes + 3.597 (F = 8.622, df = 1,2). For the nonoxygenated cardiople-
Volume 91 Number 2
Oxygenated crystalloid cardioplegia
February, 1986
26 3
20'C 15 MIN REPERFUSION AT 3l'C
,..tt-,
25
,ttt-,
20 ATP rnicromot.q"" (dry weight)
15 10
5
CONTROL
ISCHEMIC ARREST (minutes)
Fig. 1. Myocardial adenosine triphosphate (ATP) concentration after ischemic arrest at 20° C and 20 minutes of reperfusion at 37° C. Asterisks indicate differences between value indicated and preischemic (control) values whereas daggers indicate differences between groups receiving oxygenated (+02) and nonoxygenated (-0 2) cardioplegic solutions. tt **p < 0.01. ttt ***p < 0.001. Values are mean ± standard error.
20'C CARDIOPLEGIA
25 20
I3::---1 - - 1 _ _1 ...J
15 ATP micrornol.q'" (dry weight)
10
°z ° 0: I-
--I
I ***
I
(J
5 0
I
I
90
120
•
+°2
r=-o.92
N = 23
P
. -0 2
r=-o.93
N = 24
p
***-, x***
~
150
180
ISCHEMIC ARREST (minutes)
Fig. 2. Decline of myocardial ATP levels after various ischemic arrest periods at 20° C. The asterisks indicate differences for particular values indicated and control (preischemic) values. The oxygenated cardioplegia group could be characterized as: y = -0.006 (±0.002)x + 3.597 (F = 8.622, df = 1,2). The nonoxygenated group could be expressed as y = -0.021 (±0.004)x + 3.746 (F = 22.16, df = 1,2). The slopes of the two regression analyses performed differed significantly at the 5% level (F = 9.504, df = 1,4).
gia group the analysis showed the following: ATP = -0.021 (±0.004). minutes + 3.746 (F = 22.16, df= 1,2). The slopes of the two regression analyses differed at the 5% level (F = 9.504, df = 1,4). Myocardial function during reperfusion was maintained significantly better in the group receiving the oxygenated cardioplegic solution. After 120 minutes of ischemia, hearts receiving the nonoxygenated cardioplegic solution were mostly nonfunctional (Table VI). After 150 minutes none of these hearts was able to produce aortic flow. When the ATP concentration at the end of the ischemic period was used to predict postischemic func-
tion, significant correlations could be demonstrated for cardiac output (CO) (Fig. 3), peak systolic pressure (PSP) (Fig. 4), and total work (Wt). ATP/CO (r = 0.87, P < 0.001, N = 36): CO = 2.70( ±0.53) . ATP - 5.39 F = 26.36, df = 1,4 ATP/PSP (r = 0.99, P < 0.001, N = 36): PSP = 6.06( ± 0.33 . ATP - 14.82 F = 58.69, df = 1,5 ATP/Wt (r = 0.87, P < 0.001, N = 36): Wt = 0.724(±O.l4) . ATP - 3.23 F = 25.87, df = 1,4
The Journal of Thoracic and Cardiovascular
2 6 4 Coetzee et al.
Surgery
20' CARDIOPLEGIA 60
50 40 CARDIAC OUTPUT ml.mln'" 30 20 r= 0.87
10
N = 36
P <0,001
o
10
5
15
20
25
ATP mlcromol.g- 1 (dry weighl)
Fig. 3. ATP concentrations at the end of the various 20° C ischemic periods (i.e., no reperfusion) were plotted against the cardiac output attained after a 20 minute reperfusion period at 37° C. The linear regression could be expressed as: y = 2.70( ±0.53)x - 5.39 (F = 26.36, df = 1,4). 20' CARDIOPLEGIA
120 100 80 PEAK SYSTOLIC PRESSURE mmHg
60 40 20
0
/
5
r=0.99 p<0.001
10
15
20
N=36
25
ATP mlcromol.g-1 (drywelghl)
Fig. 4. ATP concentrations in the myocardium after the various 20° C ischemic arrest periods are plotted against the peak systolic pressure developed after 20 minutes of reperfusion at 37° C. The linear regression was: y = 6.06 (±0.33)x - 14.82 (F = 337.3, df = 1,5).
Electron microscopic findings. Figs. 5 and 6 are representative examples of the ultrastructure of the subepicardial and subendocardial layers of a heart subjected to 120 minutes of ischemia and nonoxygenated cardioplegia. The subepicardial layer showed considerable intramyofibrillar and intermyofibrillar edema. Mitochondria appeared rounded but otherwise normal. Sarcomeres were contracted and glycogen was absent. However, the subendocardial layer showed marked ischemic damage with severe mitochondrial swelling, clearing of the matrix, and rupture of the cristae. Rupture of mitochondrial outer membrane was seen. Sarcomeres were contracted and glycogen was absent. Hearts subjected to 180 minutes of ischemic arrest with oxygenated cardioplegia exhibited normal ultra-
structure of both layers. Fig. 7 shows a typical example of the ultrastructure of the subendocardium of such a heart. Discussion
The rationale for the use of oxygenated cardioplegic solutions is based on the need to supply oxygen to the myocardium during elective ischemic arrest periods while the subject is undergoing cardiopulmonary bypass. Use of blood cardioplegia to achieve this purpose appears to have certain disadvantages. Cold blood has a P so' that is detrimental to tissue oxygen delivery (calcu*Oxygen tension at which hemoglobin is half saturated with oxygen.
Volume 91 Number 2 February. 1986
Fig. 5. Subepicardium of a heart subjected to 120 minutes of nonoxygenated cardioplegia at 20° C. Intramyofibrillar and intermyofibrillar edema and contracted sarcomeres are present. Glycogen is absent. (Magnification X4,900.)
lated P so at 20° C = 18 mm Hg, 10° C = 15 mm Hg, and 5° C = 13.5 mm Hg I5); and since blood viscosity has an inverse relationship with temperature," oxygen delivery at a cellular level may be jeopardized. The physical laws governing oxygen flux in crystalloids predict that (1) oxygen solubility increases with a decrease in temperature, (2) all the oxygen in the solution is available for oxygen utilization, and (3) the viscosity of a crystalloid solution is affected to a lesser extent. The differences in oxygen availability from blood compared with an oxygenated crystalloid solution is evident from the following data: At 10° C an oxygenated crystalloid solution released 4.03 ± 0.07 ml oxygen/loo ml solution, which was significantly higher than the oxygen released from blood at the same temperature (hemoglobin 5.3 gm/dl, oxygen content 9.76 ± 0.48 ml oxygen/loo ml blood; oxygen released = 3.6 ± 0.1 ml/loo ml)." Therefore, blood cardioplegia cannot be accepted as the fmal answer to the issue of oxygen supply during hypothermic arrest. The feasibility of the use of an oxygenated crystalloid solution for cardioplegia was evaluated by Bodenhamer and colleagues" in 1983. In a canine heart model, these authors demonstrated a significantly improved preserva-
Oxygenated crystalloid cardioplegia
265
Fig. 6. Subendocardium of a heart subjected to 120 minutes of nonoxygenated cardioplegia at 20° C. Marked ischemic damage with severe mitochondrial swelling, clearing of the matrix, and the rupture of the cristae and outer membrane are visible. Sarcomeres are contracted and glycogen is absent. (Magnification X4,900.)
tion of myocardial function and ATP concentration after the use of an oxygenated crystalloid cardioplegic solution. Their biochemical fmdings were supported by electron microscopic data. The results obtained in our study showed that oxygenation of the cardioplegic solution at 4° C resulted in better preservation of tissue high-energy phosphates (Tables I and II). However, the differences were not reflected in subsequent myocardial function, for only at 180 minutes of ischemia were peak systolic pressures attained significantly higher in the group receiving oxygenated cardioplegia (Table V). When ATP concentrations at the end of the ischemic arrest period were correlated with cardiac output, peak systolic pressure and total work, no significant correlation could be obtained. The failure of 4°C oxygenated cardioplegia to improve functional recovery during reperfusion is probably because temperatures below 15° C are detrimental to rat hearts. Cat and rat hearts showed improved mechanical recovery when hearts were maintained at 15° C (compared with 4° C).24,25 This result is substan-
2 6 6 Coetzee et al.
Fig. 7. Subendocardium of a heart subjected to 180 minutes of oxygenated cardioplegia at 20° C. Normal ultrastructure. (Magnification X4,900.)
tiated by our finding that functional recovery of hearts subjected to 20° C oxygenated cardioplegia was significantly better than that of hearts subjected to a similar period of ischemia and 4 ° C cardioplegia (compare Tables V and VI). The lowest value for myocardial ATP content obtained at the end of an ischemic period (120 minutes of cardioplegia without additional oxygen) was 35% of the control (Group I) value. However, myocardial function during reperfusion was well preserved. These results differ slightly from those of Reibel and Revette," who reported that rat hearts with an ATP concentration less than 35% of control were unable to produce aortic output during reperfusion. Our results are clearly also at variance with the concept that reduction of ATP below 50% to 60% of control ATP is not compatible with survival of the myocardium. 3 Results obtained from our study conducted at 20° C are probably more applicable to the clinical situation. Patients at our institution who are on cardiopulmonary bypass are usually maintained at 26° C to 28° C, cardioplegic solution (at 4° C) is given every 20 minutes at a maximum, and the temperature of the operating room averages 20° C to 21° C. These circumstances suggest that myocardial temperature will be closer to 20° C than 4°C; this is especially true for the
The Journal of Thoracic and Cardiovascular Surgery
hypertrophied myocardium. In contrast to the results obtained at 4 ° C, the slope of the regression analysis for the group receiving nonoxygenated cardioplegic solution at 20° C was much steeper than for the group receiving cardioplegic solution plus additional oxygen (Fig. 2). At 180 minutes, the myocardial ATP content for the latter group did not differ significantly from the control value (Table III). The beneficial effects of the oxygenated crystalloid cardioplegic solution were also evident from the excellent preservation of myocardial microanatomy after exposure of the heart to 180 minutes of ischemia (Fig. 7). Also in this section of the protocol (20° C), it was demonstrated that a significant correlation existed between the developed peak systolic pressure, cardiac output, and total work, during reperfusion and the post-ischemic arrest ATP concentration (Figs. 3 and 4). It is therefore obviousthat the functional recoveryof the rat heart at 20° C is related to the preservation of the ATP molecules, and this observation adds weight to the argument that every possible step should be taken to allow for the maximum preservation of ATP molecules during ischemic arrest. After 90 minutes of ischemia at 20° C, hearts perfused with cardioplegic solution without additional oxygen developed acceptable peak systolic pressure, but cardiac output was reduced to approximately 50% of control values. In the period after cardiopulmonary bypass an inadequate cardiac output will lead to venous desaturation and an increase in blood lactate, a decrease in pH, and eventually circulatory decompensation. The peak systolic pressure developed after 90 minutes of ischemia may give a false impression of the cardiovascular dynamics, and we therefore suggest that oxygenation of the myocardium during a 90 minute ischemic period is as important as for 180 minute ischemic arrest periods. Our previous work showed that work performance during reperfusion was closely associated with both ultrastructure and tissue high-energy phosphate concentration." The ATP conservation that resulted from the use of oxygenated cardioplegia is reflected in the excellent myocardial functional and ultrastructural recovery after a 3 hour period of ischemia in the rat heart (Table VI, Fig. 7). Similarly, work done on canine hearts demonstrated superior recovery after 4 hours of ischemia if a crystalloid cardioplegic solution with oxygen was used." A study conducted on human beings during cardiopulmonary bypass," which used blood cardioplegia at 15° C for a mean period of 77 minutes of ischemia, demonstrated a 15% increase in ATP concentration at the end of ischemia when compared with control values.
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Oxygenated crystalloid cardioplegia
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February, 1986
Table V. Mechanical performance of rat hearts subjected to 4' C ischemic arrest followed by reperfusion at 37" C (retrograde: 10 minutes; working model: 10 minutes) Cardioplegia and reperfusion
Control (N=54) Aortic now (mi. min-I) Heart rate (beats· min-I) Peak systolic pressure (mm
I
+0,
(N= 7)
-02
+02
(N=6)
(N=5)
41.35 ±1.29 298.63 ±6.80 119.81 ±2.68
10.50** ±6.61 253.99 ±51.92 81.11 ± 17.57
24.17** ±6.21 273.70 ± 15.79 108.31 ±6.17
9.22***t ±2.04 235.50**t ± 21.70 107.60** ±4.67
15.24 ±0.55 0.51 ±0.06 15.79 ±0.51
7.58** ±2.64 0.18 ±0.15 7.76** ±2.78
9.89** ±2.05 0.15 ±0.06 10.04** ±2.09
6.80*** ± 1.06 0.15** ±0.06 6.96*** ± 1.11
I
ISO min
150 min
120 min
90 min
-02
-02
I
+02
+02
I
-02
(N=6)
(N= 11)
25.15** ±4.49 295.42 ± 29.59 94.70** ±5.65
14.88** ±4.08 240.33 ± 21.57 97.70** ±5.33
11.57*** ±3.32 264.09** ± 16.72 97.43t ±2.03
6.17*** ±2.26 279.18 ± 13.37 83.33*** ±6.35
9.44** ± 1.65 0.19** ±0.05 7.64** ±0.64
6.82** ± 1.24 0.10 ±0.03 6.92** ± 1.27
6.12*** ±0.93 0.08** ±0.03 6.20*** ±0.94
4.08*** ±0.84 0.03** ±0.01 4.10*** ±0.86
(N=5)
(N=5)
23.25** ±3.75 315.73 ± 11.46 109.95 ±3.08 10.89 ± 1.28 0.55 ±0.17 9.54** ±1.45
(N=9)
Hg) Pressure work (mW) Kinetic work (mW) Total work (mW)
l.egend: Asterisks indicate difference between values indicated and control (prcischernic) values. Daggers indicate differences between groups receiving oxygenated (+0 2 ) and nonoxygenatcd (-0 2) cardioplegia. Values are mean ± standard error.
°tp < 0.05.
"p < 0.01.
"op < 0.001.
Table VI. Mechanical performance of rat hearts after ischemic arrest at 20' C and passive reperfusion at 37" C for 10 minutes and as a working heart model for 10 minutes Cardioplegia 90 min Control (N=6)
Aortic now (mi. min-I) Heart rate (beats. min-I) Peak systolic
pressure
43.58 ± 1.39 281.00 ±2.68 109.73 ±2.98
120 min
-02
+02
(N=6)
(N=6)
11.8** ±3.42 306.58 ± 18.55 93.57** ±4.13
37.83ttt ±5.34 292.82t ±5.41 99.65*t ±3.15
I
150 min
-02
+02
(N=6)
(N=6)
1.16*** ± 1.17 107.15* ±48.18 33.80** ± 18.38 1.23*** ±0.81 om** ±0.01 1.24*** ±0.82
ISO min
-0,
+02
(N=6)
(N=6
32.90*ttt ±5.12 285.08ttt ± 10.17 106.20*ttt ±2.49
0*** 0 0*** 0 0*** 0
30.17** ±3.61 275.63 ± 15.20 90.93*** ± 15.21
12.25*ttt ± 1.14 0.14*tt ±0.04 13.81tt ±2.59
0*** 0 0** 0 0*** 0
9.55** ±0.87 0.13 ±0.05 9.63** ±0.92
I
(mm Hg) Pressure work (mW) Kinetic work (mW) Total work (mW)
14.82 ±0.86 0.21 ±0.02 15.04 ±0.88
6.03*** ±0.97 0.06** ±0.03 6.11*** ±0.98
12.00ttt ± 1.57 0.13*t ±0.03 11.05*ttt ± 1.71
Legend: Asterisks indicate differences between values indicated and (preischemic) controls. Daggers indicate differences between group receiving oxygenated cardioplegia (+0,) and hearts receiving nonoxygenated cardioplegia (-0,). Values are mean ± standard error.
otp < 0.05 . ..ttp
< 0.01.
..otttp < 0.00 I.
The 90 minute ischemic arrest period from our study at 20° C showed a 12% increase in ATP at the end of the ischemic arrest period. Although not totally comparable in terms of animals used, time perfused, and temperature used, the use of an oxygenated crystalloid cardio-
plegic solution in rats compares favorably to that of blood used in human beings. ATP levels of hearts reperfused after 90, 120,and 150 minutes of ischemia while receiving nonoxygenated 20° C cardioplegic solution remained low whereas CP
2 6 8 Coetzee et al.
content increased substantially. Similar results have been reported before in hearts reperfused after normothermic ischemic cardiac arrest. 20,27.28 The importance of oxygen supply has been highlighted in this study and confirmed the results obtained by Bodenhamer and colleagues." We suggest that this method could be used safely in patients; until the perfluorocarbon solutions are generally available," oxygenated crystalloid cardioplegia could be used to improve the ATP concentration of the myocardium during ischemic arrest. The next step in the examination of this model is the evaluation of a graded increase in the oxygen content in the crystalloid solution as well as the evaluation of the effect of additional substrate in the solution. Theoretically, the preservation of myocardial ATP, and hence function, should increase; this may extend the time for ischemic arrest during cardiopulmonary bypass even further than what has been accepted as a maximum to date. A. Bunn, Ph.D., of the Department of Medicine of the University of Stellenbosch provided assistance with the statistical analysis. REFERENCES
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Hearse DJ, Stewart DA, Chain EB: Recovery from cardiac bypass and elective cardiac arrest. Circ Res 35:448-457, 1974 Roberts AJ, Abel RM, Alonso DR, Subramanian VA, Paul JS, Gay W A: Advantages of hypothermic potassium cardioplegia and superiority of continuous versus intermittent aortic cross-clamping. J THORAC CARDIOVASC SURG 79:44-58, 1980 Bretschneider HJ, Hubner G, Knoll D, Lohr B, Nordbeck H, Spieckerman PG: Myocardial resistance and tolerance to ischemia. Physiological and biochemical basis. J Cardiavase Surg 16:241-260, 1975 Hearse DJ, Stewart DA, Braimbridge MV: Hypothermic arrest and potassium arrest. Metabolic and myocardial protection during elective cardiac arrest. Circ Res 36:481489, 1975 Hearse DJ, Stewart DA, Braimbridge MV: The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the rat. J THORAC CARDIOVASC SURG 79:39-43, 1980 Rosenfeldt FL, Hearse DJ, Cankovic-Darracott S, Braimbridge MV: The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the dog. J THORAC CARDIOVASC SURG 79:29-38, 1980 Lucas SK, Elmer EB, Flaherty JT, Prodromos CC, Buckley BH, Gott VL, Gardner TJ: Effect of multidose potassium cardioplegia on myocardial ischemia, return of ventricular function, and ultrastructural preservation. J THORAC CARDIOVASC SURG 80: 102-11 0, 1980
The Journal of Thoracic and Cardiovascular Surgery
8 Engelman RM, Anvil J, O'Donoghue MJ, Levitsky S: The significance of multidose cardioplegia and hypothermia in myocardial preservation during ischemic arrest. J THORAC CARDIOVASC SURG 75:555-563, 1978 9 Nelson RL, Fey KH, Follette DM, Livesay JJ, De Land EC, Maloney,lV, Buckberg GD: The critical importance of intermittent infusion of cardioplegic solution during aortic cross-clamping. Surg Forum 27:241-243, 1976 IO Flaherty JT, Jacobus WE, Weisfeldt ML, Hollis DL, Gardner TJ, Gott VL: Use of 31 phosphorus nuclear magnetic resonance to assess myocardial protection during global ischemia (Abstr). Am J Cardiol 43:362, 1979 II Follette DM, Mulder DG, Maloney JV, Buckberg GD: Advantage of blood cardioplegia over continuous coronary perfusion or intermittent ischemia. Experimental and clinical study. J THORAC CARDIOVASC SURG 76:604-619, 1978 12 Cunningham IN, Adams PX, Knopp EA, Bauman FG, Snively SL, Gross RI, Nathan 1M, Spencer FC: Preservation of ATP, ultrastructure, and ventricular function after aortic cross-clamping and reperfusion. J THORAC CARDlOVASC SURG 98:708-720, 1979 13 Singh A, Farrugia R, Teplitz C, Karlson KE: Electrolyte versus blood cardioplegia. Randomized clinical and myocardial ultrastructural study. Ann Thorac Surg 33:218227, 1982 14 Catinella FP, Cunningham IN, Knopp EA, Laschinger JC, Spencer FC: Preservation of myocardial ATP. Comparison of blood vs crystalloid cardioplegia. Chest 83:65065, 1983 15 Severinghaus JW: Oxyhemoglobin dissociation curve correction for temperature and pH variation in human blood. J Appl Physiol 13:485-486, 1958 16 Neely JR, Liebermeister H, Battersby EJ, Morgan HE: Effects of pressure development on oxygen consumption by isolated rat hearts. Am J Physiol 212:804-814, 1967 17 Opie LH, Mansford KRL, Owen P: Effect of increased heart work on glycolysis and adenine nucleotide in the perfused heart of normal and diabetic rats. Biochem J 124:475-490, 1971 18 Kannengieser G J, Opie LH, Van der Werff TJ: Impaired cardiac work and oxygen uptake after reperfusion of regionally ischemic myocardium. J Mol Cell Cardiol 11:197-207,1979 19 Edoute Y, Graney D, Sanan D, Kotze JCN, Lochner A: Effects of propranolol on myocardial ultrastructure, mitochondrial function and high energy phosphates of working rat hearts with coronary artery ligation. J Mol Cell Cardiol 13:619-639, 1981 20 Edoute Y, Van der Merwe E, Sanan D, Kotze JCN, Steinman C, Lochner A: Normothermic ischemic cardiac arrest in the isolated work rat heart. Circ Res 53:663-678, 1983 21 Kanter GS: Hypothermic hemoconcentration. Am J Physiol 214:856-859,1968 22 Digerness SB, Vanini V, Wideman FE: In vitro comparison of oxygen availability from asanguineous and sanguin-
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24
25
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eous cardioplegic media. Circulation 64:Suppl 2:80-83, 1981 Bodenhamer RM, DeBoer LW, Giffin G, O'Keefe D, Fallon IT, Hoetz TH, Haas GS, Daggett WM: Enhanced myocardial protection during ischemic arrest. J THORAC CARDIOVASC SURG 85:769-780, 1983 Tyers GFO, Williams EH, Hughes HC, Todd GJ: Effect of perfusate temperature on myocardial protection from ischemia. J THORAC CARDIOVASC SURG 73:766-771, 1977 Flaherty JT, Schaff HV, Goldman RA, Gott VC: Metabolic and functional effects of progressive degrees of hypothermia during global ischemia. Am J Physiol 236:H839-H845, 1979 Reibel DK, Rovetto MJ: Myocardial ATP synthesis and
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mechanical function following oxygen deficiency. Am J Physiol 234:H620-H624, 1978 27 Swain JL, Sabina RL, McHale PA, Greenfield JC, Holmes EW: Prolonged myocardial nucleotide depletion after brief ischemia in the open-chest dog. Am J Physiol 242:H818-H826, 1982 28 Kloner RA, DeBoer LWV, Darsee JR, Ingnall JS, Hale S, Tumas J, Braunwald E: Prolonged abnormalities of myocardium salvaged by reperfusion. Am J Physiol 242:H818-H826, 1982 29 Flaherty JT, Jaffm JH, Magovern GJ, Kanter KR, Gardner TJ, Miceli MV, Jacobus WE: Maintenance of aerobic metabolism during global ischemia with perfluorocarbon cardioplegia improves myocardial preservation. Circulation 69:585-592, 1984