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Ann Thorac Surg 1999;67:1871-1873
© 1999 The Society of Thoracic Surgeons

Assessing the impact of cerebral injury after cardiac surgery: will determining the mechanism reduce this injury?

^a Division of Cardiac Surgery, Johns Hopkins Hospital, Baltimore, Maryland, USA

Address reprint requests to Dr Baumgartner, Johns Hopkins Hospital, 600 N. Wolfe St., Blalock 618, Baltimore, MD 21287-4618
e-mail: wbaumgar{at}welchlink.welch.jhu.edu

Presented at the Aortic Surgery Symposium VI, April 30–May 1, 1998, New York, NY.

	Abstract

Top Abstract Introduction Material and methods Hypothesis Results Induction of nitric oxide Acknowledgments References

 
Background. Central nervous system dysfunction continues to produce significant morbidity and associated mortality in patients undergoing cardiac surgery. Using a closed-chest canine cardiopulmonary bypass model, dogs underwent 2 h of hypothermic circulatory arrest (HCA) at 18°C, followed by resuscitation and recovery for 3 days. Animals were assessed functionally by a species-specific behavioral scale, histologically for patterns of selective neuronal necrosis, biochemically by analysis of microdialysis effluent, and by receptor autoradiography for N-methyl-D-aspartate (NMDA) glutamate receptor subtype expression.

Results. Using a selective NMDA (glutamate) receptor antagonist (MK801) and an AMPA antagonist (NBQX), glutamate excitotoxicity in the development of HCA-induced brain injury was documented and validated. A microdialysis technique was employed to evaluate the role of nitric oxide (NO) in neuronal cell death. Arginine plus oxygen is converted to NO plus citrulline (CIT) by the action of NO synthase (nNOS). CIT recovery in the cerebrospinal fluid and from canine cortical homogenates increased during HCA and reperfusion. These studies demonstrated that neurotoxicity after HCA involves a significant and early induction of nNOS expression, and neuronal processes leading to widespread augmentation of NO production in the brain.

To further investigate the production of excitatory amino acids in the brain, we hypothesized the following scenario: HCA glutamate, aspartate, glycine intracellular Ca²⁺ NO + CIT. Using the same animal preparation, we demonstrated that HCA caused increased intracerebral glutamate and aspartate that persists up to 20 h post-HCA. HCA also resulted in CIT (NO) production, causing a continued and delayed neurologic injury. Confirmatory evidence of the role of NO was demonstrated by a further experiment using a specific nNOS inhibitor, 7-nitroindazole. Animals underwent 2 h of HCA, and then were evaluated both physiologically and for NO production. 7-Nitroindazole reduced CIT (NO) production by 58.4 ± 28.3%. In addition, dogs treated with this drug had superior neurologic function compared with untreated HCA controls.

Conclusions. These experiments have documented the role of glutamate excitotoxicity in neurologic injury and have implicated NO as a significant neurotoxin causing necrosis and apoptosis. Continued research into the pathophysiologic mechanisms involved in cerebral injury will eventually yield a safe and reliable neuroprotectant strategy. Specific interventional agents will include glutamate receptor antagonists and specific neuronal NO synthase inhibitors.

	Introduction

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Understanding the mechanism of cell death associated with hypothermic circulatory arrest (HCA) may provide information that is germane to all types of cardiac surgery. Central nervous system injury remains the most significant cause of morbidity in patients undergoing cardiac surgery. Neurologic dysfunction is most exaggerated in patients undergoing HCA. Newer surgical techniques such as retrograde cerebral reperfusion and innovative anesthetic techniques such as cerebrospinal fluid drainage have reduced the incidence of mortality and morbidity in this group of patients. However, the incidence of stroke, postoperative encephalopathy, and neurocognitive deficits remains problematic. These complications also strongly influence overall costs of medical care by increasing the length of stay and subsequent rehabilitation for this group of patients.

Understanding the mechanisms of central nervous system cell death associated with HCA may provide valuable information that is generic to cerebral injury involved in all types of cardiac surgery. In addition, this injury and its potential therapeutic interventional strategies may also be relevant to spinal cord injury in major aortic surgery.

	Material and methods

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For the past 7 years, our laboratory has been investigating the mechanism of cerebral injury involved in HCA. This line of investigation has used a closed cardiopulmonary bypass canine model with 2 hours of HCA followed by rewarming and resuscitation [1]. Animals underwent 20 h of intensive postoperative monitoring, after which they were extubated. They were then followed for a total of 72 hours. During that period of time, animals were assessed for functional recovery, histopathological evidence of injury, and presence or absence of specific glutamate receptors by autoradiography. Animals were assessed neurologically using a species-specific behavioral scale developed and validated for dogs at the International Resuscitation Research Center, University of Pittsburgh [2]. Histopathologic examination was performed by a single neuropathologist in a blinded manner. To assess and quantify excitatory amino acids, including glutamate and glycine, a microdialysis probe was stereotactically placed into the corpus striatum, and effluent was analyzed by high-performance liquid chromatography with electric chemical detection. In addition, citrulline (CIT), which is produced 1:1 stoichiometrically with nitric oxide (NO), was investigated.

	Hypothesis

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Neurological disturbances seen in cardiac surgery are most exaggerated in those patients undergoing HCA. In addition to computed tomography-documented stroke, these neurologic derangements can include seizures, delirium, and other manifestations of postoperative encephalopathy. Neurocognitive deficits have been reported to occur in as many as 70% of patients. The characteristic delayed neurologic sequelae after HCA in children include choreoathetosis, learning and memory deficit, and impaired IQ development.

Our research question has been: "What is the mechanism of neuronal cell injury and death in animals undergoing HCA?" Figure 1 depicts the mechanism originally proposed. We have conducted our research to validate these steps in an effort to then develop therapeutic mechanisms of intervention.

View larger version (17K): [in this window] [in a new window]   Fig 1. Proposed mechanism of neuronal cell injury and death after hypoxia and ischemia. An excessive amount of glutamate overactivates NMDA receptors. This causes an intracellular influx of calcium resulting in the production of a variety of agents, including nitric oxide synthase (NOS). This leads to the production of nitric oxide (NO), which acts as a neurotoxin, resulting in cellular death.

Glutamate receptors include the N-methyl-D-aspartate (NMDA) subtype and several non-NMDA subtypes, which are expressed in selected areas of the brain, including the hippocampus, dentate nucleus, basal ganglia, and cerebellum.

	Results

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Assessment of a glutamate receptor blocker
We wished to validate the role of excitotoxicity in a large animal model that was clinically relevant. Using the closed chest model described above, control animals were compared with a group of animals that received MK-801, which is an noncompetitive NMDA antagonist.

Dogs who received MK-801 showed minimal neurologic abnormalities compared to controls [1]. Selected neuronal necrosis was the predominant lesion observed histologically: the areas affected were those in which NMDA receptors are prominent. These include the hippocampus, cerebellum, and basal ganglia. Receptor autoradiography also demonstrated preserved NMDA receptors in animals treated with MK-801.

We felt the evidence from this study validated the concept of excitotoxicity in a clinical canine model of cardiopulmonary bypass and HCA. We have also demonstrated the efficacy of a non-NMDA receptor subtype antagonist (NBQX) in preserving neuronal receptor expression and reducing neuronal necrosis compared with control animals [5].

Assessment of the neurotransmittor glutamate
Having made the observation that a specific glutamate receptor blocker (MK-801) strongly influenced the outcome of animals undergoing HCA, we wanted to document further the role of glutamate in this process. Using microdialysis techniques, intracerebral levels of excitatory amino acids were quantitatively measured. The results were the first direct evidence that HCA causes increased intracerebral glutamate and that this increase continues up to 20 hours post-HCA [6]. In addition, there was a concomitant increase in the glutamate coagonist glycine.

	Induction of nitric oxide

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We also hypothesized that neuronal nitric oxide (NO) may be a specific neurotoxin causing neuronal cell injury and death in the same canine model of HCA. Using in vivo cerebral microdialysis, dogs were given a simultaneous infusion of artificial cerebral spinal fluid containing L-[¹⁴C] arginine or L-[¹⁴C] arginine and L-NAME (nitro arginine methyl ester), a nitric oxide synthase (NOS) inhibitor, in contralateral hemispheres during the period of HCA. L-[¹⁴C]CIT recovery, a coproduct of NO, significantly increased during HCA in the hemisphere without the inhibitor [7]. Immunocytochemical staining of the cortex with neuronal NOS-specific monoclonal antibodies (Transduction Labs, Lexington, KY) revealed increased neuronal NOS expression 6 to 18 hours after HCA. Dark-field analysis demonstrated neuronal NOS localization to neuronal processes with widespread formation of dense plexi of NOS fibers. These investigations demonstrated that neurotoxocity after HCA involves a significant early induction in neuronal NOS expression and in neuronal processes, leading to widespread augmentation of NO production in the brain.

Reduction of neuronal apoptosis using neuronal NOS inhibition
With the demonstration that neuronal NOS is induced with HCA, this study was designed to evaluate whether apoptosis, or programmed cell death, is a possible cause of the neurologic injury seen with HCA, and whether apoptosis is related to NO production. The purpose of this study, then, was to determine if neuronal NOS inhibition reduces neuronal apoptosis in the established canine model of HCA.

In this study, control dogs were compared with those treated with a neuronal NO inhibitor, 7-nitroindazole [8]. Dogs were killed at varying times from 8 to 72 hours after HCA. The degree of apoptosis was scored from 0 (normal) to 100 (severe injury). In vivo production of NOS activity was measured as CIT production. 7-Nitroindazole significantly suppressed CIT concentration compared with control animals (Fig 2). In addition, the quantitative apoptotic score in control dogs was significantly higher than in those dogs who received 7-nitroindazole treatment (61.1 ± 5.4 vs 19.2 ± 14.4; p < 0.001). Apoptosis occurred in a time-dependent fashion, peaking at 8 hours after HCA, and disappearing almost completely by 72 hours. In contrast, necrosis occurred at all three time points, but most prominently at 72 hours. Areas affected included the hippocampus, stria terminalis, neocortex, and entorhinal cortex. The NMDA receptor antagonist MK-801 was also tested in this model, and significantly reduced CIT production and apoptosis [9].

View larger version (36K): [in this window] [in a new window]   Fig 2. In vivo measurement of NOS activity as citrulline concentration (µM) over time (Reproduced with permission by Tseng EE, et al [8]).

	Acknowledgments

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This work was supported by grant 2 RO1 NS-31238-005 from the National Institutes of Health, the Dana and Albert Broccoli Center for Aortic Diseases, and The Mildred and Carmont Blitz Cardiac Research Fund.

	References

Top Abstract Introduction Material and methods Hypothesis Results Induction of nitric oxide Acknowledgments References

 

1. Redmond J.M., Gillinov A.M., Zehr K.J., et al. Glutamate excitotoxicity: A mechanism of neurologic injury associated with hypothermic circulatory arrest. J Thorac Cardiovasc Surg 1994;107:776-787.[Abstract/Free Full Text]
2. Tisherman S.A., Safar P., Radovsky A., et al. Profound hypothermia (<10°C) compared with deep hypothermia (15°) improves neurologic outcome in dogs after two hours circulatory arrest to enable resuscitative surgery. J Trauma 1991;31:1-11.[Medline]
3. Olney J.W., Ho O.L., Rhee V., et al. Neurotoxic effects of glutamate. N Engl J Med 1973;289:1374-1375.
4. Choi D.W. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1988;15:41-70.
5. Redmond J.M., Zehr K.J., Blue M.E., et al. AMPA glutamate receptor antagonism reduces neurologic injury after hypothermic circulatory arrest. Ann Thorac Surg 1995;59:579-584.[Abstract/Free Full Text]
6. Tseng E.E., Brock M.V., Kwon C.C., et al. Quantitative analyses of intracerebral excitatory amino acids and citrulline following hypothermic circulatory arrest. Surg Forum 1997;48:297-299.
7. Brock M.V., Blue M.E., Lowenstein C.J., et al. Induction of neuronal nitric oxide following hypothermic circulatory arrest. Ann Thorac Surg 1996;62:1313-1320.[Abstract/Free Full Text]
8. Tseng E.E., Brock M.V., Lange M.S., et al. Neuronal nitric oxide synthase inhibition reduces neuronal apoptosis after hypothermic circulatory arrest. Ann Thorac Surg 1997;64:1639-1647.[Abstract/Free Full Text]
9. Tseng E.E., Brock M.V., Lange M.S., et al. NMDA receptor antagonist MK-801 reduces neuronal apoptosis in a canine model of hypothermic circulatory arrest. Surg Forum 1996;47:266-269.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): William A. Baumgartner Peter L. Walinsky Jorge D. Salazar Elaine E. Tseng Malcolm V. Brock John R. Doty Michael V. Johnston Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baumgartner, W. A. Articles by Johnston, M. V. Search for Related Content PubMed PubMed Citation Articles by Baumgartner, W. A. Articles by Johnston, M. V.