Neuronal Nitric Oxide Synthase Inhibition Reduces Neuronal Apoptosis After Hypothermic Circulatory Arrest

ORIGINAL ARTICLES: CARDIOVASCULAR

Neuronal Nitric Oxide Synthase Inhibition Reduces Neuronal Apoptosis After Hypothermic Circulatory Arrest Elaine E. Tseng, MD, Malcolm V. Brock, MD, Mary S. Lange, MA, Mary E. Blue, PhD, Juan C. Troncoso, MD, Christopher C. Kwon, MS, Charles J. Lowenstein, MD, Michael V. Johnston, MD, and William A. Baumgartner, MD Division of Cardiac Surgery, Johns Hopkins Medical Institutions, and Kennedy-Krieger Research Institute, Baltimore, Maryland

Background. Neurologic injury, including choreoathetosis and learning and memory deficits, occurs after prolonged hypothermic circulatory arrest (HCA). Apoptosis, or programmed cell death, is a possible cause of the neurologic injury seen after HCA. However, the mechanism of apoptosis is unknown. Hypothermic circulatory arrest causes glutamate excitotoxicity, resulting in increased nitric oxide production. We therefore hypothesized that nitric oxide mediates apoptosis. The purpose of this study was to determine if neuronal nitric oxide synthase inhibition reduces neuronal apoptosis in an established canine model of HCA. Methods. Fourteen male hound dogs (weight, 20 to 27 kg) were placed on closed-chest cardiopulmonary bypass, subjected to 2 hours of HCA at 18°C, rewarmed to normothermia, and sacrificed 8 hours after HCA. Group 1 (n 5 7) dogs were treated with the neuronal nitric oxide inhibitor 7-nitroindazole, 25 mg/kg intraperitoneally, before arrest and every 2 hours until sacrifice. Group 2 (n 5 7) dogs received vehicle only. The brains were analyzed histopathologically. Apoptosis, identified by hematoxy-

lin-eosin staining, was confirmed by DNA terminal deoxynucleotidyltransferase–mediated dUTP-biotin nick end-labeling assay and electron microscopy. Apoptosis was scored by a blinded neuropathologist from 0 (normal) to 100 (severe injury). Results. Apoptosis occurred early after HCA in select neuronal populations, including the hippocampus, stria terminalis, neocortex, and entorhinal cortex. Apoptotic neurons showed a characteristic shrunken cytoplasm and nuclear chromatin condensation. 7-Nitroindazole significantly inhibited apoptosis (group 1 versus 2: 19.17 6 14.39 versus 61.11 6 5.41; p < .001). Conclusions. Our results provide evidence that apoptosis is associated with the neurologic injury that occurs after HCA and that nitric oxide mediates the apoptosis that occurs after HCA. Strategies for cerebral protection during HCA may include the inhibition of neuronal nitric oxide synthase.

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logic sequelae. There are two types of neuronal cell death, necrosis and apoptosis, or programmed cell death [3]. The neuropathologic hallmark of HCA has traditionally been selective neuronal necrosis of the hippocampus, neocortex, basal ganglia, and cerebellum [4 – 6]. However, the role of apoptotic cell death in HCA and its mechanism remain unknown. In previous studies in our laboratory, we have investigated the mechanism of the neuronal cell death that occurs in HCA [4 – 6]. We have shown that glutamate excitotoxicity plays a role in HCA-induced neurologic damage and that HCA causes induction of neuronal nitric oxide synthase (nNOS) [4 –7]. In this study we hypothesized that (1) apoptosis is an additional cause of the neurologic injury seen after HCA, (2) HCA results in increased nitric oxide (NO) production, and (3) NO mediates apoptosis. The purpose of this study was to characterize the nature of neuronal cell death after HCA and to determine whether nNOS inhibition reduces neuronal apoptosis in a canine model of HCA.

nitially introduced to facilitate the repair of complex congenital heart lesions, hypothermic circulatory arrest (HCA) has since been adopted for a wide array of cardiac and noncardiac procedures [1]. This technique provides a bloodless operative field, unobstructed by vascular clamps and cannulas. However, the central nervous system is most sensitive to the anoxia associated with HCA, and this therefore prohibits prolonged periods of arrest. Morbidity and mortality increase substantially if HCA lasts for more than 45 to 60 minutes [2]. Characteristic delayed neurologic sequelae seen after HCA include impaired intellectual development, choreoathetosis, and learning and memory deficits [2]. Neuronal cell death causes the HCA-induced neuro-

Presented at the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997. Address reprint requests to Dr Baumgartner, Division of Cardiac Surgery, Johns Hopkins Hospital, Blalock 618, 600 N Wolfe St, Baltimore, MD 21287 (e-mail: [email protected]).

© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

(Ann Thorac Surg 1997;64:1639 – 47) © 1997 by The Society of Thoracic Surgeons

0003-4975/97/$17.00 PII S0003-4975(97)01110-7

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Material and Methods Preparation Our canine model of HCA has been previously described [4 –7]. Fourteen conditioned, heartworm-negative, male hound dogs, 7 to 12 months old and weighing 20 to 27 kg, were used. Anesthesia was induced with sodium thiopental (3.0 mg/kg intravenously). After endotracheal intubation, dogs were maintained on halothane (0.5% to 2.0%) and 100% oxygen. Tympanic membrane, nasopharyngeal, and rectal temperature probes were placed bilaterally. The tympanic membrane temperature closely correlates with brain temperature. Electrocardiographic monitoring was employed. A Swan-Ganz catheter was placed through the left external jugular vein, and a left femoral arterial line was placed for blood pressure and arterial blood gas determinations.

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 (Sarns Inc; Ann Arbor, MI), and a 40-mm in-line arterial filter. The circuit was primed with 1.5 L of lactated Ringer’s solution, with 50 mEq of sodium bicarbonate and 10 mEq of potassium chloride. After systemic heparinization (300 U/kg intravenously), dogs were cannulated for closed-chest CPB. The arterial cannula (12F to 14F) was advanced to the descending aorta from the right femoral artery, and venous cannulas (18F to 20F) were advanced to the right atrium through the right external jugular and femoral veins. Cardiopulmonary bypass was instituted, and animals were cooled by surface (ice bags around head and cooling blanket) and core (CPB) cooling to a tympanic membrane temperature of 18°C, at which time the arterial pump was turned off and venous blood drained by gravity into the reservoir. During CPB the mean arterial pressure was maintained at 50 to 60 mm Hg with pump flows of 80 to 100 mL/kg and reduced to 60 mL/kg when the temperature was less than 32°C. Arterial blood gas values were controlled using the alpha-stat strategy. Circulatory arrest was maintained for 2 hours at 18°C, followed by the reinstitution of CPB and rewarming. Sodium bicarbonate (50 mEq) and furosemide (20 mg) were given. At normothermia (37°C), animals were weaned from CPB and decannulated. The right femoral artery and vein were ligated. Protamine was administered. Postoperatively, dogs were followed up for 8 hours after HCA. They remained on a ventilator, and anesthesia was maintained with intravenous fentanyl (10 to 20 mg/kg) and midazolam (1 mg), as needed. Intensive care unit monitoring included electrocardiography, arterial blood pressure measurements, Swan-Ganz parameters, and measurement of the urine output, as well as measurement of the arterial blood gas values, the hemoglobin concentration, and glucose levels. Animals were

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sacrificed while fully anesthetized and perfused with either ice cold saline solution or 4% paraformaldehyde. Five normal dogs that did not undergo HCA were also sacrificed and their brains harvested to serve as normal controls.

7-Nitroindazole Protocol Experimental dogs (group 1; n 5 7) received a selective nNOS inhibitor 7-nitroindazole sonicated in peanut oil at a dose of 25 mg/kg intraperitoneally before arrest, after arrest, and every 2 hours until the end of the experiment. The HCA control animals (group 2; n 5 7) received vehicle only.

Intracerebral Microdialysis Animals were positioned in a Kopf stereotactic device. The right side of the skull was exposed and burrholes placed 3 mm caudal to the coronal suture and 8 mm from the midsagittal axis. The dura mater was opened, and microdialysis probes (CMA 10/4; Acton, MA) were placed stereotactically to a depth of 20 mm into the corpus striatum. Confirmation of proper placement was determined at sacrifice by cutting the brain and visualizing the probe tract. Tissue was allowed to equilibrate for 180 minutes after probe placement. Warmed artificial cerebrospinal fluid (mmol/L concentration: NaCl, 131.8; NaHCO3, 24.6; CaCl2, 2.0; KCl, 3.0; MgCl2, 0.65; urea, 6.7; and dextrose, 3.7) was filtered and continuously bubbled with 95% oxygen and 5% carbon dioxide until the oxygen and carbon dioxide tensions were similar to those of normal brain cerebrospinal fluid. Artificial cerebrospinal fluid was infused through the inflow cannula at a rate of 1 mL/min and collected serially every 30 minutes. The effluent was assayed by high-performance liquid chromatography with electrochemical detection for extracellular amino acid concentrations [8]. The citrulline concentration was measured and used as a marker of NO production.

Histopathology Histopathologic analysis was performed in all animals. The right hemisphere of each brain was postfixed in 10% formalin and embedded in paraffin, and 8-mm sections were stained with hematoxylin-eosin. The left hemisphere was sectioned coronally, frozen on dry ice, and stored at 285°C for DNA nick end-labeling.

Electron Microscopy Coronal sections of perfused-fixed tissue were postfixed in 0.1% gluteraldehyde and 4% paraformaldehyde, osmicated, dehydrated, and embedded in epoxy resin. Ultrathin sections were cut with an ultramicrotome and examined by electron microscopy.

DNA Nick End-Labeling Apoptosis activates Ca21- and Mg21-dependent endonucleases to cut chromatin into DNA fragments. We used a terminal deoxynucleotidyltransferase (TdT)–mediated dUTP-biotin nick end-labeling (TUNEL) method (Apop Tag kit; Oncor, Inc, Gaithersburg, MD) for sensitive and

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specific staining of DNA fragmentation and apoptotic bodies associated with apoptosis.

Animal Care All experimental protocols were preapproved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions. Animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH Publications 85-23, revised 1985).

Results Time Course of Apoptosis We characterized the nature of HCA-induced neuronal cell death over time in a set of preliminary pilot experiments. A total of 21 dogs underwent 2 hours of HCA at 18°C and allowed to survive for varying lengths of time. Seven, constituting group 2, were sacrificed 8 hours after HCA, 7 at 20 hours after HCA, and 7 at 72 hours after HCA. Animals that survived for 72 hours were awakened after 20 hours and extubated. The brains were analyzed for apoptosis. Apoptosis occurred in a time-dependent fashion: it peaked at 8 hours after HCA, diminished somewhat by 20 hours, and essentially disappeared by 72 hours (Figs 1B–1D). In contrast, necrosis occurred at all three time points but most prominently at 72 hours after HCA (unpublished observations). All further experiments investigating the effects of inhibitors on apoptosis have used the 8-hour time point when apoptosis is maximal.

Histopathology identification of apoptosis. Apoptosis was identified at a morphologic and biochemical level. Electron microscopy and hematoxylin-eosin staining were used to identify apoptosis at the morphologic level, whereas the TUNEL assay was performed to assess apoptosis at the biochemical level. Morphologic criteria for apoptosis included nuclear chromatin condensation and aggregation with a peripheral distribution in the nucleus, nuclear pyknosis, cytoplasmic and cellular shrinkage, plasma membrane blebbing with no loss of integrity, fragmentation of the cell with or without nuclear fragments to produce apoptotic bodies, and the absence of an inflammatory response. Phagocytosis occurred by adjacent normal cells and some macrophages, and lysosomes remained intact. This morphologic definition remains the most consistent determinant of apoptosis. An electron micrograph of brain tissue from an animal that underwent 2 hours of HCA showed an apoptotic cell that satisfied these criteria, in contrast to a normal cell (Fig 2). Hematoxylin-eosin–stained sections at the light microscopic level also showed apoptosis, with chromatin condensation, cellular shrinkage, apoptotic bodies, and the absence of inflammation (Fig 3). At the biochemical level, apoptosis resulted in internucleosomal fragmentation of the DNA into multiples of a single-nucleosome length of DNA, approximately 180 bp.

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The TUNEL assay allowed the in situ staining of DNA fragments. Apoptosis was validated with this method (Fig 4). distribution of apoptosis. Apoptosis resulted in the deletion of single cells and was selectively distributed in neuronal subpopulations. Apoptotic cells were present in some key anatomic regions associated with memory formation. Synaptic connections of the medial temporal lobe memory system include the neocortex, entorhinal cortex, dentate gyrus, and CA regions of the hippocampus. Apoptosis occurred selectively in layer 2 of the neocortex; layers 2, 3, and 5 of the entorhinal cortex; and the dentate gyrus of the hippocampus but was not present in the CA-1 region of the hippocampus. Apoptosis rarely occurred in the bed nucleus of the stria terminalis or in the putamen and thalamus. However, of all regions, apoptosis most extensively targeted the granule cell layer of the dentate gyrus of the hippocampus, to result in near-complete destruction of this area (Figs 5A–5C).

Effect of 7-Nitroindazole on NOS and Apoptosis physiologic monitoring. Cooling times on CPB for groups 1 and 2 ranged from 25 to 30 minutes. Tympanic membrane temperatures were similar in both groups throughout the cooling, arrest, rewarming, and recovery phases (Fig 6). There were no significant differences in the esophageal and rectal temperatures between the two groups throughout the experiment. The mean arterial pressure was similar in both groups throughout the experiment, as was cardiac output. There were no significant differences in the arterial blood gas values between the two groups, with a similar pH and partial pressure of carbon dioxide. The glucose and hemoglobin levels were monitored, and both groups exhibited similar hyperglycemia and hemodilution. in vivo microdialysis. In the brain, NOS converts oxygen and arginine to NO and citrulline. Citrulline is produced 1:1 stoichiometrically with NO, and the citrulline concentration is a putative marker of NO production. The only other synthetic enzyme for citrulline, ornithine transcarbamylase in the urea cycle, is present in liver but not in brain. We measured the in vivo formation of extracellular citrulline in the basal ganglia as a function of time using a microdialysis technique. In dogs that underwent HCA alone, the citrulline concentration rose significantly above baseline (p , 0.05) during arrest (0.5 hours), during reperfusion (4 and 5 hours), and during recovery (8 hours). Treatment with 7-nitroindazole resulted in a significantly decreased citrulline concentration and therefore NO production throughout arrest, reperfusion, and at 5 and 7.5 hours during recovery (p , 0.05) (Fig 7). 7-Nitroindazole reduced the extracellular citrulline concentration by an average of 58.4% 6 28.3%. inhibition of apoptosis. Apoptosis occurred maximally in the dentate gyrus of the hippocampus. Because the

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Fig 1. Photomicrographs of hematoxylin-eosin–stained sections of dentate gyrus of hippocampus (original magnification, 3100.) (A) Normal untreated control dog. (B) Dog sacrificed 8 hours after hypothermic circulatory arrest (HCA) (group 2). Apoptotic cells are indicated with an arrow; apoptotic bodies are indicated with an arrowhead. (C) Dog sacrificed 20 hours after HCA. (D) Dog sacrificed 72 hours after HCA.

dentate gyrus was the region most amenable to quantitation of the percentage of apoptotic cells, we closely examined that region. Investigation of hematoxylineosin–stained sections revealed a reduction in apoptosis after HCA in dogs treated with 7-nitroindazole (see Fig 3). This reduction in apoptosis was confirmed by TUNEL staining (see Fig 4). Quantitatively, apoptosis was scored in a blinded fashion by a neuropathologist from 0 (normal) to 100 (severe injury), based on the percentage of cells that were apoptotic. 7-Nitroindazole significantly reduced apoptosis in dogs that underwent 2 hours of

HCA (group 1 versus 2: 19.17 6 14.39 versus 61.11 6 5.41; p , 0.001) (Fig 8).

Comment Hypothermic circulatory arrest provides a bloodless operative field, unobstructed by vascular clamps and cannulas. It is an important technique that has been widely adopted for the repair of complex congenital heart lesions, operations on the aortic arch and thoracic aorta, complex neurosurgical procedures, and excision of renal

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Fig 2. Electron micrograph of the hippocampus showing a normal cell adjacent to an apoptotic cell (arrow). An apoptotic body is indicated with an arrowhead.

cell carcinoma with vena caval or intracardiac extension [9, 10]. However, neurologic injury occurs after prolonged HCA, particularly when arrest lasts for more than 45 to 60 minutes [2]. The neurologic sequelae include seizures, choreoathetosis, learning and memory deficits, and impaired intellectual development [2, 11, 12]. Clinically apparent neurologic damage occurs in up to 12% of children and 15% of adults [2, 13]. Neuronal cell death causes HCA-induced neurologic injury. Studies investigating pharmacologic agents that can protect the central nervous system have focused on neuronal necrosis as the form of cell death induced by HCA [4 – 6]. However, the role of apoptotic cell death in HCA is not well understood. In this study, we characterized the nature of the cell death that occurs after HCA. We demonstrated that, in addition to necrosis, apoptosis plays a role in HCA-induced neurologic damage. Apoptosis occurred in a time-dependent fashion: it peaked 8 hours after HCA, diminished by 20 hours, and virtually disappeared by 72 hours. At 20 and 72 hours after HCA, substantial cell loss was noted where apoptosis had occurred, presumably stemming from the clearance of dead cells. Necrosis, in contrast, was present at 8 and 20 hours after HCA but most prominent at 72 hours. Hypothermic circulatory arrest clearly resulted in overlapping but temporally distinct phases of both apoptosis and necrosis, two distinct cell death processes. Apoptosis, or programmed cell death, is an active, tightly regulated process induced by inciting stimuli that requires energy, macromolecular synthesis, and gene transcription [3]. It results in nonrandom oligonucleosomal-length DNA fragmentation. In contrast, necrosis is a generalized failure of cellular homeostatic processes that does not require energy, macromolecular synthesis, or gene transcription [3]. It results in random DNA digestion and a loss of ion homeostatic regulation. Morphologically, apoptosis and necrosis are distinct. Apoptosis is defined by cellular shrinkage, chromatin condensation

Fig 3. 7-Nitroindazole (7-NI)–reduced apoptosis 8 hours after hypothermic circulatory arrest (HCA) as seen on hematoxylin-eosin– stained sections. Dentate gyrus at high magnification (original magnification, 3100). (A) Normal untreated control dog. (B) Dog subjected to HCA. Apoptosis is marked with an arrow; an apoptotic body is marked with an arrowhead. (C) Dog subjected to HCA treated with 7-NI.

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Fig 4. 7-Nitroindazole (7-NI)–reduced apoptosis 8 hours after hypothermic circulatory arrest (HCA) as seen on terminal deoxynucleotidyltransferase–mediated dUTP-biotin nick end-labeling–stained sections. Dentate gyrus at high magnification (original magnification, 3100). (A) Normal, untreated control dog. (B) Dog subjected to HCA. Apoptosis is marked with an arrow; an apoptotic body is marked with an arrowhead. (C) Dog subjected to HCA and treated with 7-NI.

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Fig 5. Dentate gyrus of the hippocampus from a dog sacrificed 8 hours after hypothermic circulatory arrest. (A) Photomicrograph of a hematoxylin-eosin–stained section of the entire dentate gyrus (original magnification, 34.) The structure of the dentate gyrus is noted; apoptosis is not visible at this magnification. (B) Similar magnification of dentate gyrus stained by terminal deoxynucleotidytransferase–mediated dUTP-biotin nick end-labeling (TUNEL) and viewed by dark-field microscopy. The TUNEL-positive apoptotic cells lighted up as white cells against a dark-blue background. (C) High magnification (original magnification, 340) of dentate gyrus (inset from B) viewed by bright-field microscopy. Apoptotic cells (arrows) stained dark brown, as opposed to normal cells, which stained light blue.

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Fig 6. Tympanic membrane temperatures during cooling, hypothermic circulatory arrest (HCA), rewarming, and recovery. (CPB 5 cardiopulmonary bypass; 7NI 5 7-nitroindazole.)

Fig 8. Quantitative apoptotic score of dogs that underwent hypothermic circulatory arrest (HCA) alone or HCA and 7-nitroindazole (7NI) treatment. (* 5 p , 0.001.)

and aggregation, apoptotic bodies, membrane integrity, and the absence of inflammation, whereas necrosis is characterized by cellular swelling and lysis, loss of membrane integrity, and significant inflammation [3]. With two cell death processes occurring after HCA, the question arises whether the two processes occur within the same population of neurons or are anatomically distinct. In previous studies we have investigated anatomically the selective vulnerability of neuronal necrosis [4 – 6]. Necrosis was found to occur in layers 3 and 5 of the neocortex and entorhinal cortex, the pyramidal cells of the CA-1 region of the hippocampus, the dentate gyrus of the hippocampus, the Purkinje cells of the cerebellum, and the basal ganglia (particularly the globus pallidus) [4 – 6]. In contrast, apoptosis occurred in layer 2 of the neocortex; layers 2, 3, and 5 of the entorhinal cortex; the dentate gyrus of the hippocampus; the bed nucleus of the stria terminalis; and occasionally in the thalamus and putamen (Table 1). Notably, apoptosis did not occur in

the CA-1 region of the hippocampus or in the cerebellum. Necrosis occurred in different layers of the neocortex and was much more prominent than apoptosis in the basal ganglia, whereas apoptosis predominated in the dentate gyrus. Clearly there appeared to be some anatomic specificity; however, both necrosis and apoptosis were present in the entorhinal cortex and dentate gyrus. What triggers a neuron to undergo one form of cell death versus another in response to the same inciting stimulus, that is, hypoxia and ischemia, is still unknown. To further complicate the issue, apoptosis can result in secondary necrosis if it is not cleared away by neighboring cells. Whether the necrosis in the dentate gyrus and entorhinal cortex seen at 72 hours represents primary or secondary necrosis is not known. Regardless, pharmacologic methods to reduce the neurologic sequelae after HCA must reduce both apoptosis Table 1. Selective Neuronal Injury After Hypothermic Circulatory Arrest Anatomic Location

Fig 7. In vivo measurement of nitric oxide synthase activity as citrulline concentration (mM) over time. (** 5 p , 0.05 from baseline; * 5 p , 0.05 from hypothermic circulatory arrest [HCA] control; CPB 5 cardiopulmonary bypass; 7NI 5 7-nitroindazole.)

Neocortex Layer 2 Layer 3 Layer 5 Entorhinal cortex Layer 2 Layer 3 Layer 5 Hippocampus Dentate gyrus CA-1 CA-3 Bed nucleus of stria terminalis Basal ganglia Cerebellum

Apoptosis (8 h)

Necrosis (72 h)

1 2 2

2 1 1

1 1 1

2 1 1

111 2 2 1 1 2

1 1 1 2 111 1

2 5 none; 1 5 present; 111 5 predominated.

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and necrosis. To determine ways of reducing the apoptosis, we investigated the mechanism of apoptosis in HCA. Recent evidence indicates that the mechanisms of apoptosis and necrosis after hypoxia and ischemia may be similar, with mild and intense insults resulting in apoptosis and necrosis, respectively [3, 14]. There is now an enormous amount of evidence supporting the role of glutamate excitotoxicity in hypoxic and ischemic necrosis [15], although its role in apoptosis is unclear. Hypoxia and ischemia result in an overaccumulation of the excitatory amino acid, glutamate. Glutamate stimulates Nmethyl-d-aspartate, a -amino-3-hydroxy-5-methyl-4isoxazolepropionate, and kainate glutamate receptors, to result in an increase in the intracellular calcium concentration, which then activates phospholipases, proteases, protein kinases, phosphatases, and NOS. These processes ultimately lead to protein breakdown, lipid peroxidation, and cell death [15]. Previous work in our laboratory has shown the utility of glutamate receptor antagonists in ameliorating the necrosis associated with HCA [5, 6]. Recently NO has been implicated as mediating glutamate excitotoxicity [16]. The activation of NOS by calcium entering through N-methyl-d-aspartate–type glutamate channels results in the production of NO, whose effects depend on its redox state, with NO being neurodestructive and nitrosonium ions being neuroprotective [17]. The damaging effects of NO may result from its reacting with superoxide anions to form peroxynitrite, leading to lipid peroxidation and the oxidation of sulfhydryls [17]. There is some evidence that DNA damage may be the key to NO neurotoxicity [18]. Nitric oxide triggers DNA damage, activating poly(adenosine 59-diphosphoribose) synthetase, which ultimately depletes the energy sources in the cell [18]. In this study we demonstrated that NO mediates apoptosis. Using a selective nNOS inhibitor, 7-nitroindazole, we have shown that by inhibiting nNOS, neuronal apoptosis is significantly reduced after HCA. Selective inhibition of the neuronal isoform of NOS was crucial, because we have found that the nonselective inhibition of NOS (endothelial, inducible, and neuronal) is not effective in reducing apoptosis, presumably because of a reduction in cerebral blood flow produced by endothelial NOS inhibition (unpublished observations). The reduction in the extracellular citrulline content observed in the 7nitroindazole–treated group supports the hypothesis that the drug acts by inhibiting nNOS activity. Because citrulline is generated by all three isoforms of NOS, it is possible that the 58% decrease in the citrulline concentration reflected a larger decline in the fraction contributed by nNOS alone. Although this study demonstrated the efficacy of nNOS inhibition with respect to apoptosis, further evaluation of the effect of nNOS inhibition on necrosis after HCA is warranted. Apoptosis and necrosis were both associated with neurologic injury after HCA. However, the contribution of each form of cell death to the clinical picture of neurologic damage is unknown. Necrosis predominated in the basal ganglia and cerebellum, which could result

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in uninhibited extrapyramidal activity, leading to choreoathetoid movements. Both necrosis and apoptosis occurred in the hippocampus, entorhinal cortex, and neocortex (see Table 1). Presumably either or both processes could produce the learning and memory deficits and impaired intellectual development seen after HCA, especially in children. The formation of declarative memory involves projections from the neocortex to the perirhinal and parahippocampal cortices and then to the entorhinal cortex, the major source of excitatory glutamatergic projections to the hippocampus. The entorhinal cortex subsequently projects to the dentate gyrus, CA-3, and CA-1. The implications of the selective destruction of the dentate gyrus by apoptosis or of the CA-1 region by necrosis is not known and requires further investigation. However, the intensity of apoptotic changes in the dentate gyrus is consistent with other evidence for an excitotoxic mechanism. Apoptotic cell death is important not only because of its role in physiologic neural development, but also because of its role in hypoxia, ischemia, Huntington’s disease, Alzheimer’s disease, and now the neurologic injury that occurs after HCA. Strategies for cerebral protection after HCA must ameliorate both forms of neuronal cell death, necrosis and apoptosis. The clinical safety of inhibiting nNOS, as well as its efficacy in reducing necrosis, requires further evaluation; however, it may be beneficial in reducing the severity of neurologic complications after HCA, given its ability to ameliorate apoptosis. This work was supported by The Dana and Albert Broccoli Center for Aortic Diseases and Grant 2RO1NS31238-05 from the National Institutes of Health. Elaine Tseng was supported by the Nina Braunwald Research Fellowship Award from the Thoracic Surgery Foundation for Research and Education. We thank Melissa Haggerty, Jeffrey Brawn, Chieh A. Lee, and Christopher Kwon for their technical assistance.

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DISCUSSION DR SUSUMU TANAKA (Saitama, Japan): This is an excellent and ingenious presentation, Dr Tseng. We have studied the effect of a nitric oxide synthase inhibitor on reperfusion injury of the brain after hypothermic cardiac arrest. Twelve pigs were used for this study. The NOS inhibitor l-NAME was administered intravenously just before the start of reperfusion, followed by a 60-minute continuous venous infusion. The NO concentration was determined by the electrode method. Saline solution was administered in a control group. The NO level was significantly increased in the control group, but not in the l-NAME group. Recovery of somatosensory evoked potentials was noticed in all animals in the l-NAME group but not in control pigs. These results indicate that l-NAME may protect the brain against reperfusion injury after hypothermic cardiac arrest. So I would like to ask two questions. What is the best time to administer the NOS inhibitor? Second, what is the best NOS inhibitor in humans? DR TSENG: Thank you for your comments. We administered 7-nitroindazole in this experiment before arrest, after arrest, and every 2 hours. We are also investigating the effects of nNOS inhibitors and necrosis. For those experiments, we posttreated the animals and have seen some reduction in neurologic injury. Based on our findings, either pretreatment or posttreatment is effective. With respect to humans, we feel that specific inhibition of nNOS is important. Our preliminary pilot experiments with LNNA, which also inhibits endothelial NOS, showed an increase in apoptosis. Other experimental nNOS inhibitors are being developed that may be more clinically feasible than 7nitroindazole. DR JOHN E. MAYER, JR (Boston, MA): I think this is an important issue about the different isoforms of NOS, and we probably have been on the other side of this with some experiments that show that, at least acutely, l-arginine infusions may improve the recovery of both the heart as well as cerebral blood flow after ischemia under hypothermic conditions. I wonder if you could tell us a little more about this agent and what the 7-nitroindazole effects are on the other forms of NOS, particularly inducible and endothelial forms.

DR TSENG: In vitro data indicate that 7-nitroindazole has approximately the same selectivity for neuronal and endothelial NOS but a lower selectivity for the inducible isoform. However, in vivo, the administration of 7-nitroindazole was found to have no effect on mean arterial pressure, which was confirmed in our experiments. 7-Nitroindazole in vivo acts as a relatively selective neuronal inhibitor with little endothelial effects. DR JAKOB VINTEN-JOHANSEN (Atlanta, GA): That was a very nice study. Have you done a study in which you have given l-arginine as a precursor or an NO donor to possibly augment the injury to test the opposite side of the question? Second, in your opinion, do you think that NO is working through peroxynitrite, and did you get any information or measurements on peroxynitrite production? DR TSENG: Those are very interesting questions. However, we did not use l-arginine in our model nor have we measured peroxynitrite. DR GEORGIOS KANELLOPOULOS (St. Louis, MO): Thank you for your interesting work and nice presentation. I would like to ask you what the reason was behind your choice to kill the animals in your study at 8 hours after the onset of reperfusion. I ask this question because, in studies of global brain ischemiareperfusion in the normothermic rat, neuronal apoptosis appears to be most prominent at 24 through 72 hours after the onset of the reperfusion. Therefore one might have expected that the number of apoptotic neurons in the brain would peak at 24 to 72 hours after a period of deep HCA, too. DR TSENG: Thank you for your comments. We have performed pilot experiments in which we sacrificed the animals 8, 20, and 72 hours after arrest to examine neurologic injury over time in our model. We have found that maximal apoptosis occurred at 8 hours, diminished by 20 hours, and essentially disappeared by 72 hours. This early evidence of apoptosis is unusual but may be related to the use of hypothermia, which appeared to delay the severity of necrosis to 72 hours.