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Ann Thorac Surg 1996;62:1808-1815
© 1996 The Society of Thoracic Surgeons
Division of Cardiovascular and Thoracic Surgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina
Accepted for publication July 2, 1996.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. A validated model of BD in rabbits was developed and myocardial performance was studied after BD induction and 1 hour of subsequent global hypothermic ischemia using a validated rabbit model and an isolated work-performing heart preparation.
Results. Significant decreases in stroke work, left ventricular contractility, and left ventricular relaxation were observed 2 hours after BD. After global hypothermic ischemia, significant decreases in stroke work, left ventricular contractility, and left ventricular relaxation were observed in the BD group compared with controls. Cardiac output and coronary flow were also significantly decreased in BD hearts compared with controls. Creatine kinase release was increased by 32.5% in BD hearts compared with controls.
Conclusions. In a rabbit model, BD combined with global hypothermic ischemia causes a significant decrease in left ventricular function compared with global hypothermic ischemia. This dysfunction may be attributed to a significant decrease in coronary flows in BD hearts.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The effect of brain death and global hypothermic ischemia on left ventricular function requires further investigation with particular emphasis on strategies to improve the persistent and significant incidence of early graft dysfunction after cardiac transplantation. The aims of this study were first to develop and validate a small animal model of brain death in the rabbit, and then to evaluate the added detrimental effect of hypothermic arrest on brain death associated myocardial dysfunction using an isolated, work-performing modified Langendorff apparatus to assess left ventricular function and coronary flow.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Two 22-gauge IV catheters (Gould Inc, Cardiovascular Products Division, Oxnard, CA) were placed in a large vein in each ear for maintenance IV fluids using Normosol-R, pH 7.4 (Abbott Laboratories, North Chicago, IL) at a rate of 10 mL•kg-1•h-1 and maintenance anesthesia using 7.5 µg•kg-1•h-1 fentanyl (Sigma Chemical Co, St. Louis, MO), respectively. A third catheter was placed in the femoral artery for blood pressure monitoring and arterial blood gas sampling. Lidocaine hydrochloride (Abbott Laboratories), 0.1 mg/kg, was used for arrhythmia prophylaxis, and propofol (Stuart Pharmaceuticals, Wilmington, DE), 1.5 mg/kg, was given for induction of anesthesia. Intravenous succinylcholine, 1.3 mg/kg, was given for muscle relaxation.
All animals were ventilated via tracheostomy with a 3F uncuffed endotracheal tube (Mallinckrodt Medicals, St. Louis, MO) and a Bear Infant Ventilator (Inter Med; Bear Medical Systems, Inc, Riverside, CA). The fraction of inspired oxygen was set at 1.0, the tidal volume was set at 20 mL/kg, and the rate-controlled ventilation mode was adjusted to maintain a partial pressure of arterial carbon dioxide between 35 and 40 mm Hg. The arterial pH; partial pressures of oxygen and carbon dioxide; hematocrit; levels of potassium, sodium, calcium, bicarbonate, and base excess; and percent oxygen saturation were measured (Gem-Stat; Mallinckrodt Sensor Systems, Ann Arbor, MI) at 30-minute intervals, as well as 15 minutes after any ventilator setting changes were made or medications administered. Metabolic acidosis was corrected with IV 8.4% sodium bicarbonate (Abbott Laboratories), the calcium level was balanced between 1.2 and 1.5 mmol/L with IV calcium chloride (American Regent Laboratories, Inc, Shirley, NY), and the potassium level was maintained between 4.0 and 5.0 mmol/L with IV potassium chloride (Lymphomed Inc, Deerfield, IL). Heating pads (Gaymar, Orchard Park, NY) were used to maintain a body temperature between 37° and 38°C.
Experimental Groups and Study Design
The effects of brain death and global hypothermic ischemia on left ventricular function were studied using two groups. Control hearts (n = 10) were subjected to 1 hour of global hypothermic ischemia without brain death and then studied on the modified Langendorff isolated heart apparatus. In experimental animals (n = 10), left ventricular function was assessed in a similar manner after 2 hours of brain death and 1 hour of subsequent global hypothermic ischemia.
Animals and Operation
Hearts were obtained from New Zealand white rabbits weighing 2.8 to 3.1 kg. A standard median sternotomy and anterior pericardiectomy were performed to expose the heart. After thymectomy, an ultrasonic perivascular flow probe (Transonic Systems, Inc, Ithaca, NY) was placed around the ascending aorta. Two-French Millar micromanometers (SPC 330, Millar Instruments, Inc, Houston, TX) were placed into the apex of the left ventricle and left atrium.
Induction, Diagnosis, and Validation of Brain Death
Before brain death induction, all anesthetic and paralytic agents were withheld. Brain death was induced by inflating a subdurally placed 5F Fogarty arterial embolectomy catheter (American Edwards Laboratories, Anasco, Puerto Rico) at the fusion of the frontoparietal plates with 3.0 mL of normal saline solution, causing an increase in intracranial pressure. This resulted in global cerebral and brainstem ischemia with herniation. Brain death was determined to occur when corneal and pupillary reflexes became absent after the Cushing response with cessation of spontaneous respirations.
Brain-Dead Animal Management and Organ Harvest
The experimental animals were maintained with continuous IV Normosol-R infusion at 30 mL•k-1•h-1 with routine arterial blood gas analysis every 15 minutes. No inotropic or vasoactive medications were given. After 2 hours of brain death, the hearts were harvested in the following manner. Intravenous heparin sodium (Elkins-Sinn, Inc, Cherry Hill, NJ) was given at a dose of 1,000 U/kg. The superior and inferior venae cavae were then promptly ligated using 2-0 silk suture. The hearts were vented by incising the left and right pulmonary veins, and the ascending aorta was cross-clamped distal to the origin of the coronary arteries. Cardioplegia at 4°C (10 mL/kg) was subsequently infused rapidly into the aortic root through a 20-gauge catheter. The hearts were also typically cooled with 4°C Krebs-Henseleit solution. Transection of the superior and inferior venae cavae was performed as distally as possible, and the ascending aorta was transected just proximal to the aortic cross-clamp. The left and right pulmonary veins were transected outside the pericardium. The trachea was transected at the carina, and the heart-lung block was placed in 4°C Krebs-Henseleit solution. The cardioplegic solution was based on an extracellular preparation with high osmolality (420 mOsm/L; sodium chloride, 138 mEq/L; potassium chloride, 25 mEq/L; calcium chloride, 0.7 mEq/L; magnesium chloride, 7.56 mEq/L; glucose, 15 g/L; mannitol, 20 g/L; hetastarch 6%; tromethamine to pH 7.4 to 7.5) formulated at the Duke University Medical Center Pharmacy.
Perfusion Apparatus
The standard isolated work-performing heart preparation initially described by Neely and associates [6] was modified to provide a physiologic work load equivalent to the baseline in vivo conditions (Fig 1
). A 4F inline flow probe (Transonic Systems, Inc) was connected to the aortic root cannula to measure aortic flow.
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Retrograde perfusion with oxygenated Krebs-Henseleit solution at 38.0°C was begun immediately after cannulation and deairing of the lines. Krebs-Henseleit solution was composed of (in mmol/L) NaCl, 118; KCl, 4.7; MgSO4, 7; KH2PO4 1.2; NaHCO3, 7; glucose, 11.1; CaCl2, 3; and NaEDTA, 0.5. It was bubbled with 95% oxygen and 5% carbon dioxide. Afterload pressure was maintained at 60 mm Hg to ensure adequate coronary perfusion pressure. Venting of the left ventricle during retrograde perfusion was achieved through the left atrial cannula. The coronary sinus effluent was sampled immediately at the onset of retrograde perfusion for measurement of creatine kinase levels. Millar micromanometers were placed into the apex of the left ventricle and in line with the left atrial cannula. When the coronary effluent temperatures reached 38°C, the left atrial cannula was connected to the preload reservoir and the heart was then perfused in an antegrade manner (working-heart preparation) via the left atrial cannula against an afterload of 63 mm Hg for 15 minutes before data acquisition. The isolated heart was submersed in a heated water jacket at 38°C filled with Krebs-Henseleit solution for the duration of the working mode. This preparation remained stable for 2 hours while in the working mode.
Data Acquisition and Analysis
Preload-dependent left ventricular function curves were created by raising the height of the venous reservoir in 5-mm Hg increments from 5 mm Hg up to 25 mm Hg. After 15 minutes in the working mode, the following indices of cardiac performance were measured: cardiac output, calculated as the sum of aortic and coronary flow (mL/min); heart rate (beats/min); left atrial pressure (mm Hg); left ventricular end-diastolic pressure (mm Hg); and mean developed left ventricular pressure (mm Hg). Other parameters to assess cardiac function were calculated from measured values and included the first derivative of pressure with respect to time or contractility (dP/dt, mm Hg/s), stroke volume (mL/beat), and stroke work (dyne/cm x 1,000). Stroke work was calculated by the following equation: stroke work (dyne/cm x 103) = [mean arterial pressure (mm Hg) x cardiac output (mL/min) x 1.36 mm Hg/cm H2O]/heart rate (beats/min). Coronary vascular resistance was calculated as diastolic aortic pressure - left ventricular end-diastolic pressure (mm Hg)/coronary flow (mL/min).
At the end of reperfusion, transmural left ventricular biopsy specimens were sampled for histologic analysis. The hearts were placed in a 120°C oven and dried for 24 hours. The percent of myocardial water content was calculated as 1 - (dry weight/wet weight) x 100.
All functional and hemodynamic data were digitized on-line, collected, and stored on a microprocessor (PDP 11/23; Digital Equipment Corporation, Maynard, MA). Pressure data and cardiac output were analyzed with software developed in our laboratory as described elsewhere [7]. Briefly, all data were digitized at 500 Hz and filtered by a 50-Hz low-pass filter, stored on magnetic media, and analyzed on a Zenith Z-386/20 (Zenith Data Systems Corporation, St. Joseph, MI).
Creatine Kinase Analysis
Coronary effluent taken during the first 10 minutes of retrograde reperfusion was collected in an ice-cooled beaker and stored at 0°C until analyzed. The enzyme activity was measured with a commercially available kit (Sigma Diagnostics, St. Louis, MO) using the Fiske and Subbarow procedure and a spectrophotometer (Shimadzu UV-160A, Shimadzu Corporation, Tokyo, Japan) [8]. The total amount of creatine kinase leakage was expressed as Sigma units per milliliter per gram of tissue dry weight.
Experimental Approval and Animal Care
The experimental setup and procedures conformed to the guidelines established by the American Physiological Society and the National Institutes of Health ("Guide for the Care and Use of Laboratory Animals", National Institutes of Health publication 86-23, revised 1985). The experiments were approved by the Duke University Institutional Animal Care and Use Committee (DUIACUC Assigned Registry No. A457-94-9).
Statistical Analysis
Statistical analysis was performed on an IBM personal computer using commercially available software (Sigmastat; Jandel Corporation, San Rafael, CA). First, a linear multivariate analysis of repetitive measurements was used to test an overall effect or trend over time. Because the analysis of repetitive measurements does not indicate which periods differ, follow-up period Student's t tests were used to compare mean values at baseline with data collected every 30 minutes after brain death induction. Bonferroni's method was used to compensate for the increased risk of a type I error with multiple comparisons. Reperfusion data using unpaired Student's t tests compared mean values of controls versus brain death experimental groups at each subsequent preload. Myocardial water content and creatine kinase data were also analyzed using unpaired Student's t tests comparing control with brain death. The results are expressed as mean ± standard error of the mean. A difference was considered statistically significant if the value of p was less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Two hours after brain death, significant decreases in stroke work and mean arterial pressure, as well as a significant increase in heart rate were observed in the experimental group. Positive and negative dP/dt decreased at 2 hours, but only the decrease in -dP/dt was significant (Fig 2). In addition, there were increases in cardiac output and stroke volume, although these differences did not reach statistical significance. The hemodynamic parameters for both are summarized in Table 1.
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displays the preload-dependent comparison between control and brain death hearts after 1 hour global hypothermic ischemia. Heart rates averaged from 160.1 ± 2.6 beats/min at preloads of 0 to 5 mm Hg to 174.1 ± 4.9 beats/min at preloads of 15 to 20 mm Hg for the control hearts. Brain death heart rates averaged from 161.3 ± 4.9 beats/min at preloads of 0 to 5 mm Hg to 157.1 ± 3.1 beats/min at preloads of 15 to 20 mm Hg. There were no significant differences in the heart rates between the two groups at preloads of 0 to 5 and 5 to 10 mm Hg, although heart rates were significantly less in the brain death group compared with controls (p < 0.05) at higher preloads (10 to 15 and 15 to 20 mm Hg). Stroke work was significantly greater in controls versus brain death hearts at a high preload of 15 to 20 mm Hg (59.7 ± 2.6 versus 54.5 ± 1.8 dyne/cm x 1,000; p < 0.05) (Fig 3). Coronary flow (Fig 4) was significantly decreased in the brain death hearts at low preloads (0 to 5 mm Hg) by 34.2% and high preloads (15 to 20 mm Hg) by 38.5% compared with controls (p < 0.05). At a preload of 5 to 10 mm Hg, the coronary flows in controls were greater by 34% when compared with brain death (p < 0.09). Coronary vascular resistance was increased in brain death hearts compared with controls at each preload level, reaching statistical significance at a preload of 15 to 20 mm Hg (p = 0.0048).
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Myocardial Water Content
Although there were differences in functional recovery between control and brain death hearts, there were no significant differences in myocardial water content. Control myocardial water content was 79.7% ± 1.7% in controls versus 79.8% ± 2.1% in brain death hearts.
Histologic Analysis
Histologic analysis by light microscopy revealed mild contraction band formation and interstitial edema in control hearts following reperfusion after 1 hour of global hypothermic ischemia. Brain death hearts had increased contraction band formation and myocardial necrosis as well as a similar amount of interstitial edema compared with control hearts. Coronary artery sections remained normal in both control and brain death groups.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In the present study, (1) a relatively noninvasive small animal model of brain death was established and validated in rabbits and (2) cardiac function was analyzed using a preload-dependent isolated work-performing heart apparatus to investigate the combined effect of brain death and global hypothermic ischemia on left ventricular function and coronary flow. Although this model of brain death may not exactly simulate every scenario of brain death, it does replicate the clinical findings of most patients who suffer brain death from a sudden increase in intracranial pressure caused by acute intracranial hemorrhage or head trauma [4]. By supporting the brain-dead group with only fluid and electrolyte management, we could objectively document the effects of brain death and global hypothermic ischemia on left ventricular function and coronary flow.
The results of this study clearly demonstrate that a combination of brain death and global hypothermic ischemia has a detrimental effect on left ventricular function compared with global hypothermic ischemia alone. Two hours of brain death led to a significant decline in left ventricular function in vivo. Stroke work decreased from 109.3 ± 13.5 to 58.3 ± 13.8 dyne/cm x 103 (see Table 1
), despite aggressive fluid and electrolyte management. Part of the observed hemodynamic decline was associated with peripheral vascular dilatation as cardiac output increased without any change in stroke volume 2 hours after brain death, and is consistent with the findings of both Novitzky and associates [3] and Bittner and colleagues [2]. These researchers had postulated that the mechanism of cardiac damage after brain death induction was a result of increased catecholamine levels, which induced myocyte necrosis and calcium overload [4]. Furthermore, Cooper and associates [12] demonstrated that brain death leads to a change from aerobic to anaerobic metabolism, resulting in a depletion of myocardial energy stores, which are not replenished despite anaerobic metabolism. The data from this study add to these findings in that the myocardial damage sustained during brain death appears to be further potentiated by organ preservation.
Myocardial injury after brain death in the organ donor has been studied using functional, morphologic, and biochemical analyses. Meyers and co-workers [13] demonstrated decreased myocardial blood flow after brain death experimentally using the microsphere technique. This decrease appeared to correlate with the observed impairment in left ventricular function, although there was no histologic evidence of ischemia [13].
Biochemical analyses of creatine kinase in this study revealed that lower release of postischemic enzyme was associated with better recovery of postischemic cardiac function in control hearts compared with hearts from brain-dead animals (p = 0.08) [14]. These increased creatine kinase levels support the hypothesis that brain death in combination with global hypothermic ischemia imposes further structural damage to myocardial tissue.
The postischemic dysfunction observed in the brain death hearts may be related to the significantly decreased coronary flows and increased coronary vascular resistance measured after graft preservation. Under normal resting conditions, coronary flow is dependent on heart rate and cardiac output. Heart rate did not differ significantly from controls at lower preload levels (0 to 5 and 5 to 10 mm Hg), although the coronary flows were greatly decreased by 25.5% and 25.3%, respectively, in brain death compared with control hearts (p < 0.05). Heart rate was significantly decreased in brain death compared with controls at higher preloads of 10 to 15 and 15 to 20 mm Hg (p < 0.05). One would expect increased coronary flow with decreasing heart rate as coronary flow occurs during diastole and increased time in diastole during the cardiac cycle. On the contrary, our data demonstrate that at higher preloads (15 to 20 mm Hg), heart rate was less in the brain death group compared with controls, whereas coronary flow was significantly decreased compared with control animals (p < 0.05) (see Fig 4). In addition, coronary vascular resistance in the brain death group was increased by 29.6%, 25.6%, 29.4%, and 39.1%, at increasing preload levels over the control group (p < 0.05 for preloads of 0 to 15 mm Hg and p = 0.0048 at a preload of 15 to 20 mm Hg). These data suggest that brain death and hypothermic ischemia had a significant effect on coronary vascular resistance, compared with global hypothermic ischemia alone, resulting in further ischemic injury to brain death hearts immediately after reperfusion.
Rose and associates [15] in 1988 demonstrated that brain death induction under anesthesia resulted in 9 of 11 experimental baboon hearts showing coronary arterial smooth muscle contraction bands along with focal myocardial contraction bands and myocytolytic necrosis. They concluded that coronary artery contraction bands may be useful morphologic markers for coronary spasm, although the myocardial lesions associated with increased catecholamine levels were believed to be contributed to by coronary artery spasm [15]. Novitzky and associates [4] in 1988 suggested that coronary spasm could possibly lead to transient cessation of blood flow to the subendocardial region. Histologic analysis of the rabbit myocardium demonstrated similar contraction bands in the brain death hearts, suggesting a similar injury resulting from elevated cytosolic calcium (Fig 5). These data suggest that upon reperfusion, coronary spasm may be further compromising flow to critical areas of the myocardium, thus resulting in further compromise in left ventricular function after graft preservation. The elevated coronary vascular resistance after brain death and hypothermic ischemia demonstrated in this study may be a result of coronary spasm and increased coronary artery tone after brain death as suggested by Novitzky and associates [4]. Contraction bands resulting from excessive catecholamine levels after brain death may ultimately lead to the observed coronary spasm that was apparent in this study.
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Hauptman and associates [1] demonstrated in 1994 that ischemia was a major cause of allograft dysfunction contributing to the clinical picture in at least 5 patients who suffered from early cardiac graft dysfunction. This injury may have been caused by prolonged ischemic times, poor donor preservation, or occult coronary disease in the donor hearts. They also concluded that regardless of ischemic time, poor preservation could contribute to depressed graft function. The present study has further demonstrated that hearts from brain-dead donors are more susceptible to ischemic reperfusion injury. Hauptman and associates [1] postulated that the late development of graft dysfunction may occur secondary to myocardial stunning, which clinically manifests after depletion of cellular reserve. Certainly, switching to anaerobic metabolism after brain death would decrease the normal cellular reserve of energy substrates. In the present study, we have provided additional evidence based on hemodynamic measurements that hearts harvested from brain-dead rabbits using hypothermic arrest may contain stunned myocardium, which later manifests after rewarming as ischemic myocardium due to decreased coronary flows and elevated coronary vascular resistance. This ultimately resulted in increased myocyte injury evidenced by elevated creatine kinase level measured immediately after reperfusion.
Galinanes and co-workers [16] examined the effects of leukopenic blood reperfusion on the recovery of cardiac contractile function. Their study demonstrated accelerated rate of recovery of coronary flow and contractile function with leukopenic reperfusion during the first 8 hours of reperfusion, although there was no sustained increase in the eventual extent of recovery. Others have documented the deleterious effect of neutrophils in ischemia-reperfusion injury to the heart. Intuitively, the use of crystalloid would decrease the amount of reperfusion injury and would be advantageous as compared with solution containing the various components of inflammation such as cytokines and complement. Perhaps if this study were performed with transplantation and blood reperfusion, with preload dependence, brain death would impair further ischemic reperfusion injury to the rabbit myocardium.
In summary, we have provided a valid, reproducible small animal model of brain death from which further studies concerning improvement in cardiac graft performance can be developed.
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Acknowledgments |
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Supported in part by grant HL 09315-30, awarded by the National Institutes of Health.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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