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Ann Thorac Surg 2001;72:1849-1854
© 2001 The Society of Thoracic Surgeons
a Division of Cardiac Surgery, The Johns Hopkins Medical Institutions and Kennedy-Krieger Research Institute, Baltimore, Maryland, USA
b Division of Cardiovascular Medicine, The Johns Hopkins Medical Institutions and Kennedy-Krieger Research Institute, Baltimore, Maryland, USA
c Division of Neurology, The Johns Hopkins Medical Institutions and Kennedy-Krieger Research Institute, Baltimore, Maryland, USA
d Division of Neuropathology, The Johns Hopkins Medical Institutions, and Kennedy-Krieger Research Institute, Baltimore, Maryland, USA
* Address reprint requests to Dr Baumgartner, Division of Cardiac Surgery, The Johns Hopkins Hospital, Blalock 618, 600 North Wolfe Street, Baltimore, MD 21287, USA
e-mail: wbaumgar{at}csurg.jhmi.jhu.edu
Presented at the Thirty-seventh Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 29–31, 2001.
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Abstract |
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Methods. Seventeen dogs were placed on cardiopulmonary bypass (CPB) and cooled to 18°C. After 2 hours of HCA, animals were rewarmed and weaned from CPB. Six dogs received intravenous diazoxide (2.5 mg/kg bolus 15 minutes prior to CPB, then 0.5 mg/min until circulatory arrest, then restarted for the first hour of rewarming). Six animals received vehicle only. Five received diazoxide and the mitoKATP blocker 5-hydroxydecanoate (5-HD). Using a modified Pittsburgh Canine Neurological Scoring System (0 = normal, 500 = brain death), animals were evaluated every 24 hours for 3 days. The brains were removed and histologic sections of four regions characteristically injured in this model were scored (0 = no injury, 4 = infarction) by a neuropathologist in a blinded fashion.
Results. Clinical scoring showed marked improvement in the diazoxide group at 48 hours (101 ± 10.5 vs 165 ± 14.8, p < 0.01) and 72 hours (54 ± 9.3 vs 137 ± 12.1, p < 0.01). This neuroprotection was attenuated when 5-HD was concomitantly administered. Three of four brain regions typically injured in this model (cortex, hippocampus, and entorhinal cortex) had significant neuron preservation in the diazoxide group. Likewise, combined region scores were significantly improved in the treatment group (1.18 ± 0.2 vs 2.46 ± 0.2, p < 0.01).
Conclusions. Pretreatment with diazoxide resulted in significant improvement in both clinical neurologic scores and histopathology in our model of HCA. This suggests that pharmacologic preconditioning with the mitoKATP channel opener diazoxide may offer effective neuroprotection during HCA.
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Introduction |
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Great effort has gone into investigating the exact mechanism or cascade of events leading to neuronal injury and eventual cell death. On the one hand, there is evidence for both necrotic and apoptotic (programmed cell death) pathways, with necrosis becoming the overwhelming outcome following severe, prolonged insults. On the other hand, preconditioning techniques have been successful at attenuating apoptotic injury seen with less severe insults.
Recent investigations have demonstrated a paradoxical form of protection called ischemic preconditioning (IPC) whereby brief episodes of ischemia protect against subsequent lethal ischemia [5]. In addition, pharmacologic agents that open potassium channels (PCO) on the inner mitochondrial membrane (mitoKATP) are thought to chemically produce cellular changes comparable to IPC [6].
Adenosine triphosphate (ATP)-regulated potassium channels have been identified in multiple areas of mammalian brain including within hippocampal mitochondrial membranes [7]. Recently neuroprotective conditioning has been demonstrated using mitoKATP channel openers. For example, rat neocortical brain slices encountering hypoxia-induced cell injury had complete protection against morphologic damage after pretreatment with diazoxide [8]. We sought to investigate whether pharmacologic preconditioning with diazoxide might be able to attenuate neurologic injury in a canine model of hypothermic circulatory arrest.
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Material and methods |
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Cardiopulmonary bypass and hypothermic circulatory arrest
The cardiopulmonary bypass (CPB) circuit consisted of a Cobe membrane oxygenator (Cobe Laboratories, Inc, Lakewood, CO), a Sarns roller pump system (Sarns Inc, Ann Arbor, MI), and a 40 µm arterial filter. The circuit was primed with lactated Ringer’s solution with sodium bicarbonate (50 mEq) and potassium chloride (10 mEq). After heparinization (300 U/kg IV), the right femoral artery was cannulated (12F to 14F), and the cannula was advanced into the descending thoracic aorta. Venous cannulas (18F to 20F) were advanced to the right atrium from the right femoral and jugular veins.
Closed-chest CPB was initiated, and the animals were cooled until tympanic membrane temperatures reached 18°C (about 30 minutes). Pump flows from 80 to 100 ml/kg were needed to maintain mean arterial pressures at 50 to 60 mm Hg. When the pump was turned off, venous blood was drained by gravity into the reservoir. Circulatory arrest was maintained for 120 minutes, followed by reinstitution of CPB and rewarming (about 2 hours). At 36°C the animals were defibrillated, weaned from CPB, and decannulated.
After decannulation the animals remained on the operating table, which served as an intensive care unit. Mechanical ventilation was maintained until arterial blood gases and ventilatory efforts assured successful extubation. Fentanyl (10–20 mcg/kg IV) and midazolam (1 mg IV) were used in the early postoperative period. Buprenorphine (0.3 mg IM every 12 hours for 3 days) was used for long-term pain control.
Experimental design
Diazoxide was administered as a bolus (2.5 mg/kg IV) 15 minutes before initiation of CPB, and appropriate changes in arterial blood pressure were used as an indication of blood concentration. A diazoxide infusion (0.5 mg/min) was started after administration of the bolus and continued until circulatory arrest. This infusion was again started during reinitiation of CPB and continued during the first hour of rewarming. Animals in the control group received vehicle only. A third group received a bolus of 5-hydroxydecanoate (20 mg/kg), a relatively selective mitoKATP channel blocker, at the time diazoxide was initiated. The dosage was selected in an attempt to achieve a 10:1 plasma concentration ratio to diazoxide. The drop in blood pressure seen after administration of the diazoxide bolus was due to its vascular smooth muscle relaxation properties, an attribute that that makes it a clinically available intravenous antihypertensive agent.
Neurologic scoring
All animals were neurologically scored every 24 hours for a total of 72 hours after HCA. The species-specific behavior scale used in this study was validated at the International Resuscitation and Research Center, University of Pittsburgh [13]. There were five components of neurologic function evaluated; level of consciousness, breathing pattern, cranial nerve function, motor and sensory function, and behavior. Each area was scored from 0 (normal) to 100 (severe injury) for a total from 0 (normal) to 500 (brain death).
Histopathology
All animals were killed by exsanguination under full anesthesia with perfusion of brains with 12 L of ice-cold saline at 100 mm Hg via an aortic cannula. The brains were harvested and the right hemisphere was fixed in 10% formalin, embedded in paraffin, and 8 µm sections were stained with hematoxylin and eosin and Nissl stains. The left hemisphere was sectioned, frozen with dry ice, and stored at -85°C for future use.
Sections were examined in a blinded manner by a neuropathologist (J.C.T.) for signs of neuronal necrosis or apoptosis. Criteria for apoptosis included nuclear pyknosis, cytoplasmic and cellular shrinkage, nuclear chromatin condensation and aggregation with peripheral distribution in the nucleus, fragmentation of the cell with or without nuclear fragments to produce apoptotic bodies, and the absence of inflammatory cells. Though nuclear changes consistent with apoptosis may be difficult to differentiate by light microscopy, this technique has been validated in several prior publications using TUNEL stained sections. Of particular interest were areas of the brain usually susceptible in this model of HCA; cortex (cingulate gyrus, temporal lobe, watershed region), hippocampus–dentate gyrus (dorsal, ventral), hippocampus–pyramidal cells (dorsal, ventral), entorhinal cortex, and cerebellum (Purkinje cells, granular cells). Populations of neurons were scored by the percentage of cells damaged per high-power field as follows: normal = 0, less than 25% = 1, 25%–50% = 2, 51%–75% = 3, greater than 75%, full infarct, or presence of inflammatory cells =4. The worst scoring high-power field of each region was recorded as the neuropathologist’s injury score.
Statistical analysis
Results are expressed as mean ± SEM. Comparisons between groups were made by Student’s t-test, and p values lower than 0.05 were considered significant.
Animal care
All work was preapproved by The Johns Hopkins School of Medicine Animal Care and Use Committee and performed according to the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).
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Results |
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Clinical scoring
Using the modified Pittsburgh Canine Neurological Scoring System, there was no statistical difference in clinical scoring between the diazoxide and control groups at 24 hours (p = 0.24). However, marked improvement in the treatment group developed over 48 hours and became most pronounced at 72 hours (Table 1). Devastating neurologic injury after 2 hours of circulatory arrest rendered most of the control animals severely ataxic and with profound coordination abnormalities. They were unable to sit or stand, or to perform daily tasks such as eating or drinking. In contrast, the diazoxide treatment group was uniformly approaching normal neurologic function by 72 hours; walking, eating, and sometimes jumping. If the salutary effects of diazoxide were attributable to mitoKATP channel activation, they would be expected to be antagonized by co-administration of the selective mitoKATP blocker 5-hydroxydecanoate (5-HD), a mitoKATP blocker. Indeed, the animals receiving concomitant administration of diazoxide and 5-HD had clinical scores indistinguishable statistically from control animals at 48 hours and at 72 hours (Table 1).
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Comment |
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Neuronal cell death causes HCA-induced neurologic injury. The central nervous system has the shortest "safe" period during circulatory arrest. It is generally agreed that the brain becomes more susceptible to injury as the duration of arrest increases. Postmortem histology has characterized the pathology after HCA as exhibiting a pattern of selective neuronal death. The neurons most selectively affected appear to be those in the basal ganglia, cerebellum, and hippocampus. Furthermore, these areas have been identified to be the regions that contain high concentrations of glutamate membrane receptors.
Glutamate, the most abundant free amino acid in the central nervous system, serves as a neurotransmitter that mediates signaling in excitatory pathways. Excessive accumulation of glutamate contributes to neuronal ischemic injury by overactivating neuronal receptors, precipitating a cascade of intracellular events that leads to cell death, a phenomenon termed glutamate excitotoxicity (Fig 2) [10, 15, 16]. Excessive amounts of intracellular calcium may overstimulate normal physiologic processes, activating a series of enzymes such as kinases, proteases, phosphatases, and endonuclease, and thus damaging neurons.
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Recent investigations have demonstrated an endogenous cellular protective mechanism, ischemic preconditioning, in which brief episodes of ischemia protect against subsequent lethal ischemia. Furthermore, pharmacologic agents that open ATP-sensitive potassium channels on the inner membrane of the mitochondria (mitoKATP) are thought to chemically reproduce the preconditioning and appear to alter cellular injury. This effect has been well demonstrated in the cardiovascular system [17] and has recently been shown in experimental neurologic preparations [8, 18].
The heart and brain share certain similarities: both are highly dependent on oxidative phosphorylation, are rich in mitoKATP channels, and are particularly sensitive to ischemic injury. In addition, ATP-regulated potassium channels have been identified in multiple areas of mammalian brain, including those particularly sensitive to the insults from hypothermic circulatory arrest. It is no surprise that neurons, with similar ion channels as myocytes, are afforded neuroprotection after the administration of diazoxide, previously shown to be cardioprotective. Lastly, we hypothesize that the beneficial effect of diazoxide to mitigate ischemic injury is due to this direct neuronal sparing effect, rather than secondary to cerebrovascular vasodilation; however, several mechanisms may coexist.
Possible neuroprotective mechanisms of mitoKATP openers may be occurring at several levels within neurons. MitoKATP opening may result in neuroprotection against ischemia by (1) a decrease in mitochondrial membrane potential, which decreases the driving force for lethal calcium influx; (2) "mild uncoupling," which lowers oxygen free radical production; or (3) a change in mitochondrial membrane potential, which could alter glycolytic pathways during ischemia, favoring neuronal survival. When higher, nonmitochondrial-specific drug dosing is used, other potential cellular protective effects may be recruited. Hyperpolarization of neurons through potassium channel opening on the cell membrane (plasmalemma) may result in (1) reduced excitatory amino acid release, (2) smaller increases in intracellular calcium levels, or (3) less oxygen free radical release. However, theoretical gain by this mechanism at higher drug doses may not be without the detrimental side effects of the drug.
The diazoxide dosing schedule used in this series of experiments was adapted from a protocol used for patients undergoing percutaneous transluminal coronary angioplasty in the cardiac catheterization laboratory at our institution, The Johns Hopkins Hospital. In theory, a single bolus of diazoxide prior to an ischemic insult should be adequate to initiate the cellular protective effects within the mitochondria. However, we chose to follow the successful cardioprotective protocol in our neuronal injury model, although it included an infusion of diazoxide during, and for a short time after, the insult.
In summary, we conclude that pretreatment with a selective mitoKATP opener, such as diazoxide, may provide effective neuroprotection during hypothermic circulatory arrest. This strategy could also be potentially beneficial to patients undergoing other more common cardiac surgical procedures. The mechanism attenuating neuronal injury appears to be pharmacologic preconditioning of the mitochondria, making them more resistant to future lethal insults. The exact cellular mechanism, optimal dose and delivery technique, and the safety profile, all warrant further study.
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Acknowledgments |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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2 The eleventh TSDA Resident Research Award was given to Jay G. Shake, MD, a general surgery resident, in the Department of Surgery, Johns Hopkins Hospital, Baltimore, MD. He received a monetary award of $2,500 and an engraved desktop award.
3 The TSDA, with support by Medtronic, Inc, makes this award annually, using the above selection procedure. The resident author of the selected study is recognized at the STS meeting.
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Discussion |
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DR SHAKE: Thank you, Dr Kouchoukos. Originally, I’d chosen 2 hours as a model because we had been using this preparation for roughly 8 years in our lab under Dr Baumgartner. To compare my results with previous studies, it made sense to use the same period of insult.
The 2-hour HCA period had a profound impact on our 2-hour control animals, I agree. One advantage is that we can better differentiate between the treatment and control groups if all controls are severely impaired and we then see a change in the treatment group. If a shorter insult period were used, we would likely need to do a greater number of experiments. For example, if we did a 1-hour hypothermic circulatory arrest, I would guess that our control animals would fare better, but we’d also have to have a much higher N number of experiments to find statistical significance between the groups.
DR JAKOB VINTEN-JOHANSEN (Atlanta, GA): Doctor Shake, that was a nice presentation; very good work. Did you do any scanning of the brain to look for edema, or did you measure water content of the brain? Did you look at microvascular reactivity or anything to indicate what would have been at work in producing the neurologic outcome that you observed? Further, did you use an antagonist to the mitochondrial KATP channel? Did you use an antagonist to diazoxide?
DR SHAKE: Thank you, Dr Vinten-Johansen. Those are two excellent questions.
We are certainly going to start imaging brains on our next research grant. We are pursuing novel MRI techniques to look at energetics and water content.
In response to your second question regarding a blocker to diazoxide, there is one. It is called 5-hydroxydecanoate (5-HD), and we’ve done a series of studies with that drug. The animals received diazoxide plus the blocker 5-HD. Despite adequate blocker dosages, however, we could not totally suppress the beneficial response after diazoxide administration. In fact, the animals treated with 5-HD were still significantly better than the control animals. To us, that says that there is likely a multifactorial event going on here.
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