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Ann Thorac Surg 1999;68:1578-1584
© 1999 The Society of Thoracic Surgeons
a Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
Address reprint requests to Dr Langley, Department of Cardiothoracic Surgery, Southampton General Hospital, Southampton, Hampshire, SO16 6YD, United Kingdom
e-mail: stephenlangley{at}dial.pipex.com
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
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Abstract |
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Methods. Fourteen 1-week-old piglets were randomly assigned to either placebo (n = 7), or 10 mg/kg intravenous ginkgolide B (BN52021), a naturally occurring platelet-activating factor receptor antagonist. All piglets had cardiopulmonary bypass, cooling to 18°C, 60 minutes of circulatory arrest followed by 60 minutes of reperfusion and rewarming. Global and regional cerebral blood flow, cerebral oxygen metabolism and renal blood flow were determined at baseline before DHCA and after 60 minutes of reperfusion.
Results. Blood flow was significantly reduced in all regions of the brain (p < 0.001) and the kidneys (p = 0.02) after DHCA in control animals. Cerebral oxygen metabolism was also significantly reduced after DHCA to 59.2% ± 3.2% of the pre-DHCA value (p = 0.0003). In the ginkgolide B group, recovery of global cerebral blood flow to 60.4% ± 2.8% of pre-DHCA level and of global cerebral oxygen metabolism to 77.1% ± 5.8% of pre-DHCA value were significantly higher than the recovery in the control group (p < 0.02). Regional recovery of cerebral blood flow and oxygen metabolism in the gingkolide B group was greatest in the cerebellum and brainstem. Renal blood flow did not decrease significantly after DHCA in the gingkolide B group (p = 0.23).
Conclusions. These results suggest that production of platelet-activating factor is increased in the brain after DHCA. Platelet-activating factor receptor antagonism with ginkgolide B before the circulatory arrest period can significantly improve recovery of cerebral blood flow and oxygen metabolism and renal blood flow after DHCA.
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Introduction |
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There is increasing evidence that abnormal levels of platelet-activating factor (PAF), a potent phospholipid mediator, are involved in neuronal cell damage after ischemia. Excessive PAF production occurs in pathologic states of the nervous system such as trauma, stroke, and convulsions [2]. Specific PAF receptor antagonists have been used both in the elucidation of the mechanism of PAF action and in attempts to block the effects of excess PAF production in pathologic states. One such antagonist is ginkgolide B (BN-52021), which has been used extensively in animal experiments and in clinical studies. Ginkgolide B is a naturally occurring PAF receptor antagonist that is extracted from the leaves of the ancient Chinese tree, Ginkgo biloba. Fossils of the tree date to the lower Jurassic period, 190 million years ago [3]. Ginkgo, like ginseng, is mentioned in the traditional Chinese pharmacopeia. This tree has been cultivated and held sacred in China for its health-promoting properties. Increasing experimental evidence suggests that Ginkgo biloba extracts have neuroprotective properties during cerebral ischemia, seizure activity, and against peripheral nerve damage.
The effects of PAF receptor activation are complex and incompletely defined. Stimulation of PAF receptors increases inositol phosphate production and cytosolic free calcium in cerebral tissue, an effect that can be blocked by ginkgolide B [4]. Prevention of the accumulation of intracellular calcium after ischemia is key to effective neuroprotection. Platelet-activating factor receptor activation also results in stimulation of phospholipase A2, with resulting production of arachidonic acid and eicosanoids (prostaglandins). Undoubtedly many other complex mechanisms and pathways are activated that contribute to the injury associated with excessive PAF production in neural tissue. Platelet-activating factor is a potent constrictor of cerebral arterioles in newborn pigs [5]. It also causes constriction of arterioles in other organs, an effect that can be blocked by ginkgolide B [6]. It has also been demonstrated that PAF is involved in the pathogenesis of vasospasm after subarachnoid hemorrhage. In animal models ginkgolide B and other PAF antagonists can significantly reduce selective neuronal necrosis and infarction that result from subarachnoid hemorrhage [7].
The aim of this study was to determine whether PAF is involved in the impairment of cerebral blood flow and metabolism that follows a period of DHCA. The drug ginkgolide B was chosen for these experiments because it is readily available, has an extremely low incidence of unwanted effects, and is well established as a specific PAF receptor antagonist. The null hypothesis is that PAF is not involved in the post-DHCA impairment in cerebral blood flow (CBF) and cerebral metabolism (CMRO2) and that ginkgolide B given before cardiopulmonary bypass (CPB) will have no effect on cerebral recovery after 60 minutes of DHCA in the neonatal piglet.
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Material and methods |
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Fourteen neonatal piglets (1 to 2 weeks old; weight, 2.5 ± 0.1 kg, mean ± standard error of the mean) were anesthetized with an intramuscular injection of ketamine (50 mg/kg) and acepromazine (15 µg/kg). Intravenous methylprednisolone (30 mg/kg) was administered through a 24-guage cannula in the marginal vein of the pinna. Following orotracheal intubation, mechanical ventilation (Infant Ventilator, Sechrist Industries, Anaheim, CA) was commenced to achieve arterial oxygen tensions of 150 to 250 mm Hg and carbon dioxide tensions of 35 to 45 mm Hg. The animals were paralyzed with intravenous pancuronium (300 µg/kg) and anesthetized with fentanyl (100 µg/kg). Thereafter, anesthesia was maintained with a continuous infusion of fentanyl (25 µg/kg per hour). An 18-guage cannula was placed in the descending aorta via the femoral artery for blood pressure monitoring and arterial blood sampling. The animal’s temperature was monitored throughout the study by an indwelling nasopharyngeal temperature probe (Yellow Springs Instrument Inc, Yellow Springs, OH). Temperature was maintained at 36°C except for the period of induced hypothermia.
The heart was exposed through a median sternotomy. Cardiac instrumentation consisted of a 3-F micromanometer (Millar Instruments Inc, Houston, TX) inserted into the superior vena cava for central venous pressure monitoring and placement of an 8-mm flow probe (Transonic Systems, Ithaca, NY) around the proximal pulmonary artery for cardiac output monitoring.
Sagittal sinus access
The animals were anticoagulated with intravenous heparin (500 IU/kg) before access of the sagittal sinus. A 1-cm strip of scalp was raised in the midline over the vertex of the skull. Two separate 2-mm burr holes were made over the superior sagittal sinus for repeated sagittal sinus venous blood sampling and continuous sagittal sinus venous pressure monitoring with a 3-F micromanometer (Millar Instruments Inc, Houston, TX).
Cardiopulmonary bypass and circulatory arrest
An 8-F arterial cannula and a 20-F venous cannula (DLP Inc, Grand Rapids, MI) were inserted through purse-string sutures into the ascending aorta and the right atrium, respectively. Cardiopulmonary bypass was commenced at a flow rate of 120 mL/kg per minute. The pump-oxygenator system consisted of a Sarns nonpulsatile roller pump (Sarns Inc, Ann Arbor, MI) and a Medtronic Minimax PLUS hollow fiber membrane oxygenator (Medtronic Inc, Anaheim, CA). No arterial filter was used. The circuit was primed with heparinized fresh blood from a donor pig. Ringer’s lactate and sodium bicarbonate solutions were added to the priming solution to achieve a hematocrit of 0.25 and a pH of 7.4 at 37°C. The total prime volume was approximately 450 mL. The temperature of the perfusate was controlled with the integral heat exchanger in the venous reservoir of the oxygenator and a BIO-CAL 370 water bath system (Biomedicus, Minneapolis, MN). Animals were cooled to a temperature of 18°C over a standard duration of 20 minutes by the circulation of ice water through the heat exchanger. At the end of the cooling period, the circulation was arrested, and the animal drained. DHCA was established and the aortic and right atrial cannulas were clamped. After 60 minutes of DHCA, the aortic and venous cannulas were unclamped. Perfusion was reestablished at 120 mL/kg per minute with the perfusate initially at room temperature (20° to 22°C). Rewarming was accomplished by circulating warm water to the heat exchanger in the venous reservoir. A nasopharyngeal temperature of 36°C was generally reached by 45 minutes of reperfusion. During cooling and rewarming, blood gases were managed according to the alpha-stat strategy. The arterial pH was maintained at 7.35 to 7.45 and carbon dioxide tension at 35 to 45 mm Hg measured at 37°C and uncorrected for the temperature of the animal. Arterial oxygen tension was kept between 150 and 250 mm Hg and hematocrit between 0.23 and 0.28. Sodium bicarbonate (8.4%) was given when necessary but not immediately before cerebral blood flow measurements. At the end of the study, the animals were killed by a bolus injection of fentanyl and cessation of CPB.
Measurement of cerebral blood flow
Cerebral blood flow measurements were determined by the reference-sample, radiolabeled microsphere technique [8] during CPB at 36°C. The technique in the current study was identical to that used in previous reports from our laboratory [9]. Suspensions of microspheres with a diameter of 15.5 ± 0.1 µm (DuPont de Nemours & Co, Wilmington, DE) were made up in 10% dextran and 0.01% TWEEN 80 with 106 microspheres per milliliter. Two different isotopes were used in each piglet and these were chosen at random from the three different isotopes available (scandium 46, ruthenium 103, and niobium 95). For each flow measurement, 106 microspheres were injected into a side port of the arterial tubing 30 cm proximal to the aortic cannula over 30 seconds and washed through with 5 mL warm saline solution. A reference blood sample was withdrawn from the distal aorta at a constant rate of 3 mL/minute with a Harvard syringe pump (Harvard Apparatus, South Natick, MA), commencing 10 seconds before the microsphere injection and continuing for a total of 2 minutes. At the end of the experiment, the brain was removed and divided into left and right cerebral hemispheres, basal ganglia, cerebellum, and brain stem (midbrain, pons, and medulla oblongata). The kidneys were also removed for determination of renal blood flow (RBF). After measurement of fresh weights, the brain parts and kidneys were dissolved in 2 mol potassium hydroxide solution and analyzed, together with the reference blood sample, in a gamma counter (Auto-Gamma 5530; Packard Instrument Co, Meriden, CT) to estimate the quantity of each type of radiolabeled microsphere present in the specimen. The withdrawal rate of the reference blood sample and the ratio of counts from a brain part to the reference blood sample allowed calculation of regional cerebral blood flow. Cerebral blood flow measurements are expressed in milliliters per 100 grams of brain per minute by normalizing for fresh tissue weight. The weighted sum of regional cerebral blood flow allowed calculation of global cerebral blood flow.
Cerebral perfusion pressure was taken as the difference between the mean arterial pressure and the sagittal sinus venous pressure. Cerebral vascular resistance was the ratio of cerebral perfusion pressure to global cerebral blood flow (in units of mm Hg/100 g per minute per milliliter). Systemic vascular resistance was taken as the ratio between the systemic perfusion pressure and the total bypass pump flow rate (systemic vascular resistance = [mean arterial pressure - right atrial pressure]/[pump flow rate] in units of mm Hg/100 g per minute per milliliter).
Measurement of cerebral oxygen handling
Arterial and sagittal sinus blood samples were taken just before each microsphere injection for estimation of oxygen tension, carbon dioxide tension, oxygen saturation, pH, and base excess by using a GEM-Stat Blood Gas/Electrolyte Monitor (Mallinckrodt Sensor Systems Inc, Ann Arbor, MI). Hemoglobin levels (in g/dL) were measured from arterial blood samples (482 Co-Oximeter; Instrumentation Laboratory Corp, Lexington, MA). Cerebral delivery of oxygen (CDO2 in milliliters per 100 grams of brain per minute), cerebral metabolic rate of oxygen (CMRO2 in milliliters per 100 grams of brain per minute), and cerebral oxygen extraction (CEO2 as a percent) were calculated as follows: CDO2 = cerebral blood flow x arterial oxygen content; CMRO2 =cerebral blood flow x (arterial oxygen content - sagittal sinus venous oxygen content); and CEO2 = (CMRO2/CDO2) x 100%. The oxygen content (in units of mL of O2 per mL of blood) was calculated by the following formula: O2 content = 0.01 x [(1.36)(hemoglobin)(oxygen saturation) + (0.003)(oxygen tension)].
Experimental protocol and data collection
The animals were randomly assigned to two groups with 7 animals in each group. The control group received 1 mL alcohol (alcohol-anhydrous: 95% ethanol, 5% isopropanol; Eastman Kodak Co, Rochester, NY) intravenously at induction plus 1 mL in the pump. The study group, GINKGO B, received 10 mg/kg ginkgolide B (Sigma Chemical Company, St Louis, MO) in 1 mL alcohol intravenously at induction plus an additional 10 mg/kg in 1 mL alcohol in the pump. All animals were cannulated for CPB and normothermic perfusion was commenced at 120 mL/kg per minute. The animals were stabilized on normothermic CPB for a minimum of 20 minutes before the pre-DHCA measurement was taken. During this time the pump flow was adjusted to provide a constant cerebral perfusion pressure of 50 mm Hg. After the pre-DHCA measurement, pump flow was returned to 120 mL/kg per minute, and the animals were cooled for DHCA. After 60 minutes of DHCA circulation was recommenced and the animals rewarmed. At 45 minutes of reperfusion, pump flow rate was again adjusted to provide a cerebral perfusion pressure of 50 mm Hg for 15 minutes before the post-DHCA cerebral blood flow measurement was taken at 60 minutes of reperfusion. Data collected at the two time points included nasopharyngeal temperature, mean arterial blood pressure, right atrial pressure, sagittal sinus venous pressure, arterial and sagittal sinus blood gases, CPB flow rate, electrolytes, hematocrit, hemoglobin, and cerebral and renal blood flow.
Statistical analysis
All the results were entered into an Excel 97 spreadsheet (Microsoft Corporation, Redmond, WA) for further analysis. Repeating formulas calculated the mean and standard error of the mean for all the data collected in each animal. Further repeating formulas calculated the CBF, CDO2, CEO2, CMRO2, and the percentage change between each variable before and after DHCA for all animals. A two-tailed, paired-samples t test was used to compare means at different time points within a group. An unpaired (independent samples) t test was used to compare means between the groups. Statistical significance was tested at the 95% confidence limit.
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Results |
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Effects of deep hypothermic circulatory arrest on the brain
Sixty minutes of DHCA in the control group resulted in a decrease in the systemic vascular resistance (SVR) from 0.44 ± 0.02 mm Hg/100 g per minute per milliliter pre-DHCA to 0.36 ± 0.02 mm Hg/100 g per minute per milliliter (p < 0.02). This decrease necessitated an increase in mean pump flow from 131 ± 7 mL/kg per minute before DHCA to 164 ± 10 mL/kg per minute after DHCA (p = 0.03) to maintain the preset cerebral perfusion pressure of 50 mm Hg at the time of CBF measurement. Cerebral blood flow was significantly reduced in all regions of the brain after DHCA (p < 0.001) (Table 1). Different brain regions were affected to different degrees, with lowest recovery of blood flow in the cerebral hemispheres (40.4% ± 2.6% of pre-DHCA level) and greatest in the cerebellum (69.0% ± 4.3% of the baseline) (Fig 1).
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Global cerebral oxygen handling in the ginkgolide B group is shown in Figure 4. The global CDO2 was significantly lower after DHCA in the ginkgolide group than before the arrest period (p = 0.0005) (Table 2). The mean global CDO2 decreased to 62.3% ± 4.0% of the pre-DHCA level, which was a significantly greater than the 50.4% ± 3.0% recovery in control animals (p = 0.03). In the ginkgolide B group, after DHCA there was a significant reduction in CDO2 in the cerebral hemispheres and the basal ganglia compared with that before DHCA (p = 0.0005). However, in the cerebellum and brain stem there was no significant difference in CDO2 after DHCA (p = 0.2). The percentage recovery was again greatest in the cerebellum (92.1% ± 4.9% of baseline) and lowest in the cerebral hemispheres (57.3% ± 5.4% of baseline). In all regions the recovery of CDO2 was significantly greater in the ginkgolide group compared with controls (p < 0.05).
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Comment |
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In the animals that received ginkgolide B the SVR increased significantly after DHCA. The increase in SVR with PAF antagonism compared with the control group suggests that the decrease in SVR in control animals was due to vasodilatory effects of PAF. The pump flow in the ginkgolide B group was reduced to maintain the preset level of cerebral perfusion pressure at 50 mm Hg. After DHCA, CBF and CDO2 were significantly greater in the ginkgolide B group than in the control group in all brain regions. Platelet-activating factor is known to reduce CBF [11]; similarly, PAF administration can cause a dose-dependent decrease in spinal cord blood flow [12]. Ginkgolide B has been shown to improve CBF after reperfusion following an ischemic insult and to inhibit maturation of ischemic injury [13]. Platelet-activating factor antagonists (including ginkgolide B), however, do not alter normal CBF or cerebral oxygen consumption [14], which suggests that, under normal circumstances, levels of PAF do not modulate CBF or metabolism and antagonists to PAF receptors might therefore have a wide safety margin. The reduced cerebral vascular resistance with gingkolide B compared with the control group accounts for an improvement in CBF. Part of this PAF-induced cerebral vasoconstriction is due to release of vasoactive arachidonic acid metabolites, which can be blocked by indomethacin [15]. Recovery of CDO2 occurred to such an extent in the cerebellum and brainstem that there was no significant difference in regional levels before and after DHCA, which reflects the relatively higher CBF to these regions. After DHCA, the CMRO2 was also significantly better in the ginkgolide B group.
In the brain there is considerable evidence that abnormal levels of PAF are involved in neuronal cell damage after ischemia and inflammation. Ginkgolide B can modulate the cerebral response to ischemia [16]. It has a neuroprotective effect on primary neuronal cultures isolated from the cerebral cortex [17] and reduces postischemic histologic damage in the hippocampus [18]. Antagonism of PAF also reduces both the size of cerebral infarction and the resulting motor deficit [19]. In perinatal hypoxia-ischemia, ginkgolide B decreases the incidence and severity of brain injury [20]. Furthermore it has recently been shown that PAF has an important role in glutamate neurotoxicity [17].
The proposed mechanism involves a PAF-mediated release of cerebral cellular lipids and free fatty acids, resulting in increased cerebral edema and cell injury. Brain edema is one of the most important clinical complications in ischemic brain damage. It is well established that PAF increases vascular permeability in various circulatory beds [21], including cerebral arteries, a phenomenon inhibited by PAF antagonists. With regard to neuronal injury, phospholipase A2 mediates accumulation of free fatty acid in the brain, and ginkgolide B is thought to reduce activation of this enzyme [22]. Platelet-activating factor stimulates release of arachidonic acid and enhances thromboxane B2 (the stable thromboxane A2 metabolite) production in the brain. Ginkgolide B has been shown to block thromboxane B2 synthesis completely [23].
It has been known for 10 years that ginkgolide B protects the kidney against ischemic injury [24]. The current study found improved renal blood flow in the ginkgolide group after DHCA. This is certainly pertinent to the use of CPB and DHCA in children, where impaired renal function postoperatively can cause serious morbidity. In addition, postoperative pulmonary edema is reduced by PAF antagonism. Previous studies have shown a reduction in pulmonary injury after CPB with the use of a PAF antagonist [25]. As well as the potential benefits for neurologic protection, PAF antagonism during CPB could contribute to improved renal and pulmonary function.
Platelet-activating factor is of considerable importance in central nervous system physiology and pathology, and administration of PAF receptor antagonists can be beneficial during cerebral ischemia. A more precise understanding of the mechanisms underlying these effects, including the seemingly complex cross talk between PAF and other mediators, could lead to the clinical use of PAF receptor antagonists to interrupt the PAF-biochemical cascade that leads to cell death. The improvement in early cerebral recovery from a period of DHCA with ginkgolide B is encouraging. It suggests that PAF is responsible for at least some of the cerebral impairment that follows DHCA. Antagonism of PAF receptors resulted in an improvement in CBF and cerebral metabolism, and the null hypothesis stated earlier can be rejected. Potentially PAF antagonism can also prevent initiation of some of the complex pathways and mechanisms that lead to ischemic neuronal damage. Although it remains to be seen whether ginkgolide B reduces long-term neurologic damage, this study gives an encouraging start to the use of PAF receptor antagonists in the context of DHCA.
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Acknowledgments |
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no significant difference from pre-DHCA value within group, paired t test p > 0.05.)
