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Ann Thorac Surg 1999;67:65-71
© 1999 The Society of Thoracic Surgeons

Original Articles

Nitric oxide mediates neurologic injury after hypothermic circulatory arrest

Elaine E. Tseng, MDa, Malcolm V. Brock, MDa, Mary S. Lange, MAa, Juan C. Troncoso, MDa, Charles J. Lowenstein, MDa, Mary E. Blue, PhDa, Michael V. Johnston, MDa, William A. Baumgartner, MDa

a Division of Cardiac Surgery, Johns Hopkins Medical Institutions and Kennedy-Krieger Research Institute, Baltimore, Maryland, USA

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

Presented at the Forty-fourth Annual Meeting of the Southern Thoracic Surgical Association, Naples, FL, Nov 6–8, 1997.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Prolonged hypothermic circulatory arrest (HCA) causes neurologic injury. However, the mechanism of this injury is unknown. We hypothesized that HCA causes nitric oxide production to result in neuronal necrosis. This study was undertaken to determine whether the neuronal nitric oxide synthase inhibitor 17477AR reduces necrosis after HCA.

Methods. Thirty-two dogs underwent 2 hours of HCA at 18°C. Nitric oxide synthase catalytic assay and intracerebral microdialysis for nitric oxide production were performed in acute nonsurvival experiments (n = 16). Sixteen animals survived for 72 hours after HCA: Group 1 (n = 9) was treated with 17477AR (Astra Arcus), and group 2 (n = 7) received vehicle only. Animals were scored from 0 (normal) to 500 (coma) for neurologic function and from 0 (normal) to 100 (severe) for neuronal necrosis.

Results. Administration of 17477AR reduced nitric oxide production in the striatum by 94% (HCA alone), 3.65 ± 2.42 µmol/L; HCA and 17477AR, 0.20 ± 0.14 µmol/L citrulline). Dogs treated with 17477AR after HCA had superior neurologic function (62.22 ± 29.82 for group 1 versus 141.86 ± 61.53 for group 2, p = 0.019) and significantly reduced neuronal necrosis (9.33 ± 4.67 for group 1 versus 38.14 ± 2.23 for group 2, p < 0.00001) compared with untreated HCA dogs.

Conclusions. Our results provide evidence that neuronal nitric oxide synthase mediates neuronal necrosis after HCA and plays a significant role in HCA-induced neurotoxicity. Pharmacologic strategies to inhibit neuronal nitric oxide synthase after the ischemic period of HCA may be clinically beneficial.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Hypothermic circulatory arrest (HCA) enables a bloodless operative field, unobstructed by vascular clamps and cannulas. It facilitates repair of aortic arch and thoracic aortic lesions in addition to complex congenital heart lesions [1–3]. However, the brain is exquisitely sensitive to ischemia and prohibits lengthy periods of arrest. Morbidity and mortality increase substantially when the duration of HCA exceeds 45 to 60 minutes [4]. Impaired intellectual development, choreoathetosis, and learning and memory deficits are characteristically delayed neurologic sequelae after prolonged HCA [5–7].

Previous studies in our laboratory have shown that glutamate excitotoxicity plays a role in HCA-induced neurologic damage [8–10]. Recent evidence suggests that nitric oxide (NO) mediates glutamate excitotoxicity [11]. In addition, we previously showed that HCA causes induction of neuronal nitric oxide synthase (nNOS) and that inhibition of nNOS reduces apoptosis, one form of neuronal cell death after HCA [12, 13]. In the present study, we hypothesized that HCA results in increased NO production and that NO mediates ischemic necrosis. Our purpose was to determine whether nNOS inhibition reduces neuronal necrosis in a canine model of HCA.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Preparation
Our canine model of hypothermic circulatory arrest has been described elsewhere [12, 13]. Thirty-two heartworm-negative conditioned male hound dogs (weight, 20 to 27 kg; age, 7 to 12 months) were used. Anesthestic induction with sodium thiopental (3.0 mg/kg intravenously) followed by endotracheal intubation was performed. Animals were maintained with halothane (0.5% to 2.0%) anesthesia and 100% oxygen.

Bilateral tympanic membrane, nasopharyngeal, and rectal temperature probes were placed. Tympanic membrane temperature closely correlates with brain temperature. Electrocardiographic monitoring was used. A Swan-Ganz catheter was placed percutaneously through the left external jugular vein, and a left femoral artery catheter was placed for blood pressure and arterial blood gas determinations.

Cardiopulmonary bypass and hypothermic circulatory arrest
The cardiopulmonary bypass (CPB) circuit consisted of a membrane oxygenator (Cobe Laboratories, Inc, Lakewood, CO), roller pump (Sarns Inc, Ann Arbor, MI), and 40-µm 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. An arterial cannula (12F to 14F) was placed in the descending aorta from the right femoral artery, and venous cannulas (18F to 20F) were advanced to the level of the right atrium through the right external jugular and femoral veins.

Cardiopulmonary bypass was instituted, and the dogs were cooled by surface (ice bags around the head and a 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 was drained by gravity into the reservoir. During CPB, mean arterial pressure was maintained at 50 to 60 mm Hg with pump flows of 80 to 100 mL/kg and was reduced to 60 mL/kg when the temperature was less than 32°C. Arterial blood gas levels were controlled using the alpha-stat strategy.

Circulatory arrest was maintained for 2 hours at 18°C, followed by reinstitution of CPB and rewarming. Sodium bicarbonate (50 mEq) and furosemide (20 mg) were given. At normothermia (37°C), dogs were weaned from CPB and decannulated. The right femoral artery and vein were ligated. Protamine was administered. Wounds were irrigated and closed.

Postoperatively, 16 dogs were monitored for 8 to 20 hours after HCA and were sacrificed for acute experiments. Ventilation was continued, and anesthesia was maintained with intravenous fentanyl (10 to 20 µg/kg) and midazolam (1 mg) as needed. The remaining 16 animals survived for 72 hours and were weaned from the ventilator at 20 hours. The electrocardiogram, arterial blood pressure, Swan-Ganz catheter variables, and urinary output, as well as arterial blood gas, hemoglobin, and glucose levels were monitored in the intensive care unit. Animals were sacrificed fully anesthetized and perfused with either ice-cold saline or 4% paraformaldehyde. Five normal dogs that did not undergo HCA were also sacrificed, and their brains were harvested for normal control values.

17477AR protocol
Experimental dogs (group 1, n = 9) received the selective nNOS inhibitor 17477AR (Astra Arcus USA, Rochester, NY) as a 1.5-mg/kg intravenous infusion for 30 minutes (0.5 mg/mL in lactated Ringer’s solution titrated to pH 4.0) after HCA at decannulation and every 12 hours for 24 hours postoperatively. The half-life of 17477AR’s ability to inhibit nitric oxide synthase (NOS) activity was approximately 12 hours, according to Astra Arcus. The HCA control animals (group 2, n = 7) received vehicle only.

Intracerebral microdialysis
Intracerebral microdialysis was performed in 16 nonsurvival experiments animals that subsequently underwent HCA. Eight dogs were used to measure inhibition of NO production by 17477AR; the remaining 8 served as the HCA control group. Animals were positioned in a Kopf stereotactic device. The right side of the skull was exposed, and burr holes were placed 3 mm caudal to the coronal suture and 8 mm from the midsagittal axis. The dura was opened, and microdialysis probes (CMA 10/4; Acton, MA) were stereotactically placed to a depth of 20 mm into the corpus striatum. Confirmation of proper placement was determined at sacrifice by brain cutting and visualization of the probe tract. Tissue was allowed to equilibrate after probe placement for 180 minutes. Warmed artificial cerebrospinal fluid (mmol/L concentration: sodium chloride, 131.8, sodium bicarbonate, 24.6, calcium chloride, 2.0, potassium chloride, 3.0, magnesium chloride, 0.65, urea, 6.7, and dextrose, 3.7) was filtered and continuously bubbled with 95% nitrogen and 5% carbon dioxide until oxygen and carbon dioxide tensions were similar to those of normal brain cerebrospinal fluid. Artificial cerebrospinal fluid was infused through the inflow cannula at 1 µL/min and serially collected every 30 minutes. Effluent was assayed by high-performance liquid chromatography with electrochemical detection for extracellular amino acid concentrations [14, 15]. Citrulline concentration was measured and used as a marker of NO production. All concentrations were not corrected for probe efficiency.

Nitric oxide synthase assay
Cortical tissues were collected, homogenized, and assayed for NOS activity, as described by Bredt and Snyder [16]. This assay measured the amount of carbon-14 (14C) arginine converted to 14C-citrulline by calcium-dependent NOS.

Clinical neurologic injury evaluation
Surviving dogs were neurologically assessed every 12 hours for 72 hours after HCA, according to a species-specific behavior scale developed and validated for dogs at the International Resuscitation Research Center, University of Pittsburgh [17]. Five components of neurologic function were evaluated, including level of consciousness, breathing pattern, cranial nerve function, motor and sensory function, and behavior; each was scored from 0 (normal) to 100 (severe injury), for a total of 0 (normal function) to 500 (brain death).

Histopathologic analysis
Histopathologic analysis was performed in all surviving dogs at 72-hours after HCA. The right hemisphere of each brain was post-fixed in 10% formalin and embedded in paraffin, and 8-µm sections were stained with hematoxylin and eosin or cresyl violet. The left hemisphere was sectioned coronally, frozen on dry ice, and stored at -85°C for NOS assay.

Hematoxylin and eosin–stained sections of 25 anatomic regions were examined in blinded manner by a neuropathologist (J.T.) for neuronal necrosis. Each population of neurons was scored as follows: normal = 0; few ischemic neurons = 1; moderate ischemic neurons = 2; infarct with loss of neurons = 3; and hemorrhagic infarct = 4. Neuronal populations were then divided into six anatomically related regions for comparison between experimental groups of dogs. Total histopathologic score for each brain was the sum of all scores for each of 25 anatomic regions, ranging from 0 (normal) to 100 (severe injury).

Statistical analyses
Results are expressed as mean ± standard deviation of the population. Comparisons between groups were made by analysis of variance for repeated measures or Student’s t test where appropriate.

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" published by the National Institutes of Health (NIH Publications No. 85-23, revised 1985).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Intracerebral microdialysis
In the brain, NOS converts oxygen and L-arginine to NO and citrulline in a 1:1 ratio. Because the only other synthetic enzyme for citrulline—ornithine transcarbamylase in the urea cycle—is present in the liver but not in the brain, citrulline concentration can be used as a marker of NO production. We used microdialysis to measure in vivo formation of extracellular citrulline in the basal ganglia as a function of time. In dogs undergoing HCA alone, peak citrulline concentration rose significantly (p < 0.05) above baseline during reperfusion CPB and 2 to 8 hours after HCA (Fig 1). Treatment with 17477AR at 2 hours after HCA resulted in a significantly decreased citrulline concentration and therefore a reduced NO production 2 to 8 hours after HCA (p < 0.05) (Fig 1). Administration of 17477AR reduced peak extracellular citrulline to 0.20 ± 0.14 µmol/L compared with 3.65 ± 2.42 µmol/L for HCA alone and 1.01 ± 1.06 µmol/L at baseline. Mean arterial pressure was not affected by administration of 17477AR.



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  		Fig 1. In vivo measurement of NOS activity as citrulline concentration (µmol/L) over time. Peak levels of citrulline were plotted over three time periods: HCA, reperfusion CPB 2 to 8 hours after HCA. (* = p < 0.05, 2 to 8 hours versus baseline. ** = p < 0.05, 17477AR versus untreated HCA control [Ctl] group.)

 
Nitric oxide synthase assay
Nitric oxide synthase assay measures tissue NOS activity by the amount of 14C-citrulline produced from 14C-arginine over time. There are three forms of NOS; inducible NOS is calcium independent, whereas endothelial and nNOS are calcium dependent. Nitric oxide synthase assay in the presence of calcium was performed in the dorsolateral neocortex. Nitric oxide synthase activity was significantly reduced in dogs treated with 17477AR at 72 hours after HCA compared with NOS activity in untreated HCA dogs at 72 hours (group 1 versus group 2: 0.497 ± 0.217 versus 4.138 ± 0.064 x 10-12 moles 14C-citrulline formed per milligram of protein per minute, p < 0.05) or untreated control dogs (group 1 versus untreated control dogs: 0.497 ± 0.217 versus 4.552 ± 0.62 x 10-12 moles 14C-citrulline formed per milligram of protein per minute, p = 0.0157) (Fig 2). The nNOS inhibitor 17477AR had a long half-life of approximately 12 hours and when administered 24 hours after HCA, had lingering effects at 72 hours. Untreated control dogs were normal dogs sacrificed without CPB or HCA.



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  		Fig 2. Nitric oxide synthase (NOS) activity (pmol 14C-citrulline · mg protein-1 · min-1) in the dorsolateral neocortex in untreated control dogs, HCA dogs at 72 hours, and 17477AR-treated dogs at 72 hours after HCA.*p < 0.05 compared with untreated HCA dogs.

 
Physiologic monitoring
Cooling times during CPB for both groups ranged from 25 to 30 minutes. Tympanic membrane temperatures were similar for both groups throughout cooling, arrest, rewarming, and recovery phases (Fig 3). No statistically significant differences were found between the two groups to explain the cerebroprotection of 17477AR. There were no significant differences in esophageal and rectal temperatures between the two groups throughout the experiment. Mean arterial pressure was similar in both groups throughout the experiment, notably even with 17477AR administration, as was cardiac output. There were no significant differences in arterial blood gases between the two groups, with similar pH and carbon dioxide tension. Glucose and hemoglobin levels were monitored, and both groups exhibited similar hyperglycemia and hemodilution.



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  		Fig 3. Tympanic membrane temperatures (°C) during cooling CPB, HCA, rewarming CPB, and recovery.

 
Neurologic outcome
At each 12-hour neurologic evaluation after HCA, dogs treated with the nNOS inhibitor 17477AR had significantly better neurologic deficit scores than untreated HCA dogs (Fig 4). Final neurologic scores were 62.22 ± 29.82 for the group treated with 17477AR and 141.86 ± 61.53 for the untreated HCA group (p = 0.019).



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  		Fig 4. Neurologic deficit score of HCA dogs versus 17477AR-treated dogs over time. (* = p < 0.05, 17477AR versus HCA alone.)

 
Histopathologic analysis
Hypothermic circulatory arrest resulted in selective neuronal necrosis in layers 3 and 5 of the entorhinal cortex, basal ganglia, Purkinje cells of the cerebellum, layers 3 and 5 of the neocortex, and hippocampus, most notably the pyramidal cells of CA-1. Neuronal necrosis was significantly less in 17477AR-treated dogs than HCA dogs, most notably in the neocortex, hippocampus, cerebellum, and basal ganglia (Fig 5). Total histopathologic score for the 17477AR-treated group was 9.33 ± 4.67 versus 38.14 ± 4.67 (p < 0.00001) for the untreated HCA group (Table 1).



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  		Fig 5. Treatment with 17477AR reduced necrosis 72 hours after HCA, as seen by hemotoxylin and eosin–stained sections of the CA1 region of the hippocampus (40x magnification): (A) normal untreated control dog; (B) HCA dog at 72 hours (arrows indicate necrosis); (C) dog treated with 17477AR after HCA.

 

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  		Table 1. Histopathologic Score for Each Regiona

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Initially introduced for the repair of complex congenital heart lesions, HCA has been adopted for aortic arch and thoracic aortic procedures [1–3]. However, the brain is the most sensitive organ to ischemia and prohibits prolonged periods of arrest. Neurologic injury occurs after prolonged HCA when the duration of arrest exceeds 45 to 60 minutes [4]. Clinical neurologic sequelae include seizures, choreoathetosis, learning and memory deficits, and impaired intellectual development, which occurs in up to 12% of children and 15% of adults [4–7].

Neuronal cell death causes HCA-induced neurologic injury. Selective neuronal necrosis is the hallmark of neuropathologic injury after HCA. Much evidence now supports the role of glutamate excitotoxicity in mediating hypoxic and ischemic neuronal necrosis [18]. Hypoxia and ischemia result in overaccumulation of the excitatory amino acid glutamate. Extracellular glutamate stimulates glutamate receptors, including N-methyl-D-aspartate, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate receptors, to result in a rise in intracellular calcium [18]. Calcium then activates phospholipases, protein kinases, proteases, and NOS and triggers a cascade of cellular events that leads to lipid peroxidation, DNA degradation, and eventually neuronal cell death. We previously showed that glutamate receptor antagonists, including N-methyl-D-aspartate and {alpha}-AMPA receptor antagonists, reduced HCA-induced neuronal necrosis [8–10].

Recently, NO has been implicated in mediating glutamate excitotoxicity [11, 19–21]. In transgenic mice missing the neuronal NOS gene, cerebral infarcts from middle cerebral artery occlusion were significantly smaller in size than control mice [21]. Activation of NOS by calcium entering through the N-methyl-D-aspartate receptor channels results in increased NO, which may be neuroprotective or neurodestructive, depending on its redox state [19]. Nitrosonium ion may be neuroprotective by reacting with sulfhydryl groups on the N-methyl-D-aspartate receptor, whereas NO may be neurodestructive by reacting with superoxide anion to form peroxynitrite. Peroxynitrite causes lipid peroxidation and oxidation of sulphydryls [19]. In addition, NO can damage DNA by base deamination and activate poly(adenosine 5'-diphosphoribose) polymerase (PARP) to repair damaged DNA. Activation of PARP may then lead to progressive cell depletion of adenosine triphosphate and death [20].

Nitric oxide has beneficial effects in other tissues, such as the heart, during ischemia and reperfusion, because of its endothelial-relaxing factor effect of vasodilatation. This aspect may also be beneficial during reperfusion injury in the brain to increase cerebral blood flow; however, it is counteracted by the neurotoxic effects of NO, hydroxyl radical, nitrogen dioxide, and peroxynitrite.

We previously showed that nNOS was induced after HCA and that nNOS inhibition reduced one form of neuronal cell death (apoptosis) after HCA [12, 13]. In the present study, we hypothesized that HCA causes NO production and that NO or its metabolites, or both, cause neuronal necrosis. We examined citrulline production as a marker for NO production over time. Citrulline production increased significantly over baseline during reperfusion CPB and at 2 to 8 hours after HCA. We administered 17477AR after reperfusion CPB and found peak citrulline production significantly reduced by 94% compared with HCA alone. Administration of 17477AR reduced basal NO production by 80%.

In addition, calcium-dependent NOS activity in neocortical tissues was determined. There are three forms of NOS: calcium-independent inducible NOS, calcium-dependent endothelial NOS, and nNOS. Previously, we showed that calcium-independent NOS activity was negligible compared with calcium-dependent NOS activity [12]. In addition, we also demonstrated by immunohistochemical analysis that only nNOS, not endothelial or inducible NOS, was induced by HCA [12]. Because calcium-dependent NOS activity predominated, and 17477AR did not inhibit endothelial NOS in vivo, as seen by its lack of elevation of mean arterial pressure, nNOS was most likely responsible for the increases in NO seen after HCA. In the present study, calcium-dependent NOS activity 72 hours after HCA was unchanged from that in normal dogs at baseline; however, 17477AR treatment significantly reduced nNOS activity compared with nNOS activity in untreated control and HCA dogs at 72 hours.

Microdialysis experiments determined increases in NOS activity in the caudate over time, specifically during and early after HCA, but was not measured as late as 72 hours. Nitric oxide synthase assay revealed no changes in NOS activity in the neocortex at fixed time points of sacrifice at 8, 20, and 72 hours after HCA. The lack of increase in NOS activity by NOS assay as opposed to high-performance liquid chromatographic assay most likely reflects two causes. Differences may be explained by different intracerebral locations where activity was measured and by the ability of one technique to measure activity over time as opposed to the fixed time points, when the activity returned to baseline.

Anatomically, HCA demonstrated selective vulnerability of neurons to necrosis. Necrosis occurred in layers 3 and 5 of the neocortex and entorhinal cortex, the pyramidal cells of CA-1 hippocampus, dentate gyrus of the hippocampus, Purkinje cells of the cerebellum, and the basal ganglia. We demonstrated that NO mediated necrosis. Using the selective nNOS inhibitor 17477AR, we showed that nNOS inhibition significantly reduced neuronal necrosis when given after HCA. Reduction in extracellular citrulline observed in the 17477AR-treated group supported the hypothesis that the drug acted through inhibition of nNOS activity. Reduction in necrosis was most notable in the cerebellum, hippocampus, neocortex, and basal ganglia.

Clinically, no adverse effects of 17477AR administration were seen. Dogs treated with 17477AR demonstrated superior neurologic function with regard to level of consciousness, breathing pattern, cranial nerve function, motor and sensory function, and behavior compared with dogs that underwent HCA alone.

Notably, reduction in neurologic injury was demonstrated despite administration after the ischemic period of arrest. A number of reasons may explain this outcome. First, NO production increased significantly during reperfusion CPB and recovery. Unlike glutamate, which is released immediately during ischemia, NO production is further downstream in the cascade. In cell cultures, this downstream effect may still be manifested as in conjunction with glutamate release. However, NO production requires oxygen and in the present in vivo system, prolonged HCA is essentially an oxygen deprivation model. Although some NO production is likely to occur during ischemia, the bulk of it occurred during reperfusion and thereafter, suggesting a beneficial effect of post-HCA administration. Second, nNOS is also located along arterioles and is believed to play a role in regulation of cerebral blood flow; notably, inhibition of nNOS may reduce cerebral blood flow. Post-HCA CPB reperfuses the brain, reintroducing oxygen and nutrients; reduction of cerebral blood flow during this initial reperfusion period may be harmful to neurons in the ischemic penumbra. The beneficial effects of nNOS inhibition may depend on a careful balance between cerebral blood flow and cellular toxic NO production. For these reasons, we chose to inhibit nNOS after reperfusion CPB. Because NO production continued in a delayed fashion after HCA, we administered 17477AR for up to 24 hours postoperatively.

Of note, inhibition of nNOS reduced neurologic injury; however, the relative contribution of reduction of NO versus hydroxyl radical, nitrogen dioxide, and peroxynitrite production could not be determined in the present study. Inhibition of nNOS presumably prevented further downstream toxic effects of NO and its metabolites. Ultimately, the cause of direct tissue injury at the cellular level was not determined in the present study but is most likely due to peroxynitrite, formed by reaction of NO with superoxide anion. However, we did demonstrate that inhibition of nNOS successfully blocked the neurotoxic downstream cascade and reduced neuronal necrosis.

We previously showed that both apoptosis and necrosis were associated with neurologic injury after HCA [13]. Hypothermic circulatory arrest results in problems with declarative memory. Declarative memory involves projections from the neocortex to perirhinal and parahippocampal cortices and then to the entorhinal cortex. The entorhinal cortex, a major source of glutamate pathways to the hippocampus, subsequently projects to the dentate gyrus, CA-3, and CA-1. Both necrosis and apoptosis occurred in the hippocampus, entorhinal cortex, and neocortex [13]. Either or both processes could explain impaired intellectual development and the learning and memory deficits seen after HCA. Hypothermic circulatory arrest also causes choreoathetosis. Necrosis predominated in the basal ganglia and cerebellum, which could result in uninhibited extrapyramidal activity, leading to choreoathetoid movements. We previously showed that apoptosis is reduced by nNOS inhibition. In the present study, we demonstrated reduction of neuronal necrosis by 17477AR. Inhibition of nNOS significantly reduced both forms of neuronal cell death—apoptosis and necrosis—and clinically resulted in improved neurologic function. Clinical safety of nNOS will require further evaluation; however, it may be beneficial in reducing neurologic complications even when given after HCA.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
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 technical assistance. We also thank David Reif and Astra Arcus, USA for providing 17477AR and its pharmacokinetic data.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Niazi S.A., Lewis F.J. Profound hypothermia: report of a case. Ann Surg 1957;147:264-266.
  2. Coselli J.S., Buket S., Djukanovic B. Aortic arch operation: current treatment and results. Ann Thorac Surg 1995;59:19-27.[Abstract/Free Full Text]
  3. Kouchoukos N.T., Daily B.B., Rokkas C.K., Murphy S.F., Bauer S., Abboud N. Hypothermic bypass and circulatory arrest for operations on the descending thoracic and thoracoabdominal aorta. Ann Thorac Surg 1995;60:67-77.[Abstract/Free Full Text]
  4. Kirklin J.W., Barratt-Boyes B.G. Hypothermia, circulatory arrest, and cardiopulmonary bypass. In: Kirklin J.W., Barratt-Boyes B.G., eds. Cardiac surgery. New York: Churchill Livingstone, 1993:66-73.
  5. Newburger J.W., Jonas R.A., Wernovsky G., et al. A comparison of the perioperative neurologic effects of hypothermic circulatory arest versus low-flow cardiopulmonary bypass in infant heart surgery. N Engl J Med 1993;329:1057-1064.[Abstract/Free Full Text]
  6. Bellinger D.C., Jonas R.A., Rappaport L.A., et al. Developmental and neurologic status of children after heart surgery with hypothermic circulatory arrest or low-flow cardiopulmonary bypass. N Engl J Med 1995;332:549-555.[Abstract/Free Full Text]
  7. Davis E.A., Gillinov A.M., Cameron D.E., Reitz B.A. Hypothermic circulatory arrest as a surgical adjunct: a 5-year experience with 60 adult patients. Ann Thorac Surg 1992;53:402-407.[Abstract]
  8. Redmond J.M., Gillinov A.M., Blue M.E., et al. The monosialoganglioside, GM1, reduces neurologic injury associated with hypothermic circulatory arrest. Surgery 1993;114:324-333.[Medline]
  9. 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]
  10. 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]
  11. Dawson T.M., Snyder S.H. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 1994;14:5147-5159.[Abstract]
  12. Brock M.V., Blue M.E., Lowenstein C.J., et al. Induction of neuronal nitric oxide after hypothermic circulatory arrest. Ann Thorac Surg 1996;62:1313-1320.[Abstract/Free Full Text]
  13. 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]
  14. Carlberg M. Assay of neuronal nitric oxide synthase by HPLC determination of citrulline. J Neurosci Methods 1994;52:165-167.[Medline]
  15. Donzanti B.A., Yamamoto B.K. An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates. Life Sci 1988;43:913-922.[Medline]
  16. Bredt D.S., Snyder S.H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990;87:682-685.[Abstract/Free Full Text]
  17. Tisherman S.A., Safar P., Radovsky A., et al. Profound hypothermia (<10°C) compared with deep hypothermia (15°C) improves neurologic outcome in dogs after two hours circulatory arrest to enable resuscitative surgery. J Trauma 1991;31:1-11.[Medline]
  18. Lipton S.A., Rosenberg P.A. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 1994;330:613-622.[Free Full Text]
  19. Lipton S.A., Choi Y.B., Pan Z.H., et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitrosos-compounds. Nature 1993;364:626-631.[Medline]
  20. Zhang J., Dawon V.L., Dawson T.M., Snyder S.H. Nitric oxide activation of poly (ADP)-ribose synthetase in neurotoxicity. Science 1994;263:687-689.[Abstract/Free Full Text]
  21. Huang A., Huang P.L., Panahian N., Dalkara R., Fishman M.C., Moskowitz M.A. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 1994;265:1883-1885.[Abstract/Free Full Text]



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G. Amir, C. Ramamoorthy, R. K. Riemer, V. M. Reddy, and F. L. Hanley
Neonatal Brain Protection and Deep Hypothermic Circulatory Arrest: Pathophysiology of Ischemic Neuronal Injury and Protective Strategies
Ann. Thorac. Surg., November 1, 2005; 80(5): 1955 - 1964.
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T. Zaitseva, G. Schears, S. Schultz, J. Creed, D. Antoni, D. F. Wilson, and A. Pastuszko
Circulatory Arrest and Low-Flow Cardiopulmonary Bypass Alter CREB Phosphorylation in Piglet Brain
Ann. Thorac. Surg., July 1, 2005; 80(1): 245 - 250.
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C. Mavroudis and R. M. Sade
The Southern Thoracic Surgical Association 50th anniversary celebration: the impact of STSA pediatric cardiothoracic surgery manuscripts on surgical practice
Ann. Thorac. Surg., November 1, 2003; 76(90050): S47 - 67.
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Y. Cho, T. Ueda, A. Mori, T. Nakamichi, H. Shimizu, Y. Inoue, and S. Kawada
Exogenous aspartate neurotoxicity in the spinal cord under metabolic stress in vivo
Ann. Thorac. Surg., November 1, 2000; 70(5): 1496 - 1500.
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T.-A. Miyamoto and K.-J. Miyamoto
Only redox state of cytochrome a,a3 reflects adequacy of tissue oxygenation
Ann. Thorac. Surg., May 1, 2000; 69(5): 1642 - 1643.
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T. A. Miyamoto and K.-J. Miyamoto
Would pH-stat strategy generate nitric oxide during hypothermic perfusion?
Ann. Thorac. Surg., January 1, 2000; 69(1): 312 - 313.
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E. E. Tseng, M. V. Brock, C. C. Kwon, M. Annanata, M. S. Lange, J. C. Troncoso, M. V. Johnston, and W. A. Baumgartner
Increased intracerebral excitatory amino acids and nitric oxide after hypothermic circulatory arrest
Ann. Thorac. Surg., February 1, 1999; 67(2): 371 - 376.
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