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Ann Thorac Surg 2006;81:1593-1598
© 2006 The Society of Thoracic Surgeons

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Original article: Cardiovascular

Noninvasive Assessment of Brain Injury in a Canine Model of Hypothermic Circulatory Arrest Using Magnetic Resonance Spectroscopy

^a Division of Cardiac Surgery, Kennedy-Krieger Research Institute, Baltimore, Maryland
^b Division of Neurology, Kennedy-Krieger Research Institute, Baltimore, Maryland
^c Division of Neuropathology, Kennedy-Krieger Research Institute, Baltimore, Maryland
^d Department of Radiology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Accepted for publication January 4, 2006.

^_* Address correspondence to Dr Baumgartner, Division of Cardiac Surgery, The Johns Hopkins Medical Institutions, 600 North Wolfe St, Blalock 618, Baltimore, MD 21287 (Email: wbaumgar{at}csurg.jhmi.jhu.edu).

Presented at the Poster Session of the Fifty-second Annual Meeting of the Southern Thoracic Surgical Association, Orlando, FL, Nov 10–12, 2005.

	Abstract

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
BACKGROUND: Studies have confirmed the neuroprotective effect of diazoxide in canines undergoing hypothermic circulatory arrest (HCA). A decreased N-acetyl-asparate:choline (NAA:Cho) ratio is believed to reflect the severity of neurologic injury. We demonstrated that noninvasive measurement of NAA:Cho with magnetic resonance spectroscopy facilitates assessment of neuronal injury after HCA and allows for evaluation of neuroprotective strategies.

METHODS: Canines underwent 2 hours of HCA at 18°C and were observed for 24 hours. Animals were divided into three groups (n = 15 in each group): normal (unoperated), HCA (HCA only), and HCA+diazoxide (pharmacologic treatment before HCA). The NAA:Cho ratios were obtained 24 hours after HCA by spectroscopy. Brains were immediately harvested for fresh tissue NAA quantification by mass spectrometry. Separate cohorts of HCA (n = 16) and HCA+diazoxide (n = 23) animals were kept alive for 72 hours for daily neurologic assessment.

RESULTS: Cortical NAA:Cho ratios were significantly decreased in HCA versus normal animals (1.01 ± 0.29 versus 1.31 ± 0.23; p = 0.004), consistent with severe neurologic injury. Diazoxide pretreatment limited neurologic injury versus HCA alone, reflected in a preserved NAA:Cho ratio (1.21 ± 0.27 versus 1.01 ± 0.29; p = 0.05). Data were substantiated with fresh tissue NAA extraction. A significant decrease in cortical NAA was observed in HCA versus normal (7.07 ± 1.9 versus 8.54 ± 2.1 µmol/g; p = 0.05), with maintenance of normal NAA levels after diazoxide pretreatment (9.49 ± 1.1 versus 7.07 ± 1.9 µmol/g; p = 0.0002). Clinical neurologic scores were significantly improved in the HCA+diazoxide group versus HCA at all time points.

CONCLUSIONS: Neurologic injury remains a significant complication of cardiac surgery and is most severe after HCA. Magnetic resonance spectroscopy assessment of NAA:Cho ratios offers an early, noninvasive means of potentially evaluating neurologic injury and the effect of neuroprotective agents.

	Introduction

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Hypothermic circulatory arrest (HCA) is a commonly employed neuroprotective strategy for complex congenital heart operations and for procedures involving the great vessels. Although profound hypothermia provides reasonable neurologic protection, significant neurologic sequelae, including stroke, movement disorders, and impairment of memory and learning, can occur, especially when the arrest time exceeds 45 minutes [1]. These clinical deficits are attributed to neuronal injury and death in various regions of the brain [2, 3].

The development of potential therapeutic strategies is dependent on our understanding of the mechanism of neuronal cell injury after cardiac surgery. "Glutamate excitotoxicity" is one major mechanism of neuronal injury that can lead to neuronal hyperactivity and death during periods of metabolic stress such as ischemia and hypoxia [4, 5]. Glutamate initiates a cascade of events through binding to an N-methyl-D-aspartate receptor, ultimately resulting in neuronal necrosis or apoptosis. Histologically, the areas of the brain most significantly affected are those in which N-methyl-D-aspartate receptors are prominent: the hippocampus, cerebellum, and basal ganglia [6]. Pharmacologic blockade of this receptor has been shown to provide some degree of neurologic protection in animal models, thus validating this mechanism of injury [7].

Nitric oxide is a ubiquitous molecule that can act as a neurotoxin when present in excessive amounts. Induction of neuronal nitric oxide synthase occurs downstream from N-methyl-D-aspartate receptor activation and leads to widespread nitric oxide production in the brain [8, 9]. Nitric oxide and its metabolite peroxynitrite have toxic effects on the neuronal mitochondria, resulting in free radical production and DNA fragmentation. This leads to mitochondrial energy failure, which plays a central role in neuronal cell death. Previous investigations using an animal model of HCA have shown that inhibition of neuronal nitric oxide synthase resulted in decreased nitric oxide production and superior neurologic function [6].

Many different pharmacologic agents have been explored for their potential neuroprotective effects in both experimental and clinical models. The inhibition of mitochondrial energy failure is an important strategy to prevent ischemia-induced neuronal injury. Ischemic preconditioning is a paradoxical form of protection against lethal ischemia in which cells are exposed to brief episodes of conditioning ischemia before primary insult. Although initially described in cardiac myocytes, a similar mechanism has also been demonstrated in neurons, potentially providing neuronal mitochondrial protection. One potential mechanism of ischemic preconditioning relies on the opening of adenosine triphosphate–dependent K⁺ channels on the inner mitochondrial membrane [10, 11]. This effect can be achieved pharmacologically using a variety of different agents. Diazoxide, which was initially used as an antihypertensive medication, is one such adenosine triphosphate–dependent K⁺ channel agonist. Pretreatment with diazoxide before neurologic insult has been associated with improved functional outcomes and histopathology [12]. These studies have demonstrated that pharmacologic intervention at specific points in the injury cascade may potentially mitigate the neurologic deficits that can result from cardiac surgery.

The evaluation of postoperative neurologic injury is an area of intensive investigation. Studies have assessed the accuracy of various methods including neuropsychologic testing, CNS molecular markers, and magnetic resonance imaging. However, these techniques have limited sensitivity and an inadequate ability to prognosticate long-term neurologic outcomes. Proton magnetic resonance spectroscopy (MRS) is a technology that uses metabolite information from various brain regions to permit sensitive, noninvasive assessment of neurochemical alterations after brain injury. This information can aid in determining clinical neurologic prognosis [13]. One such metabolite, N-acetyl-aspartate (NAA), which is ubiquitous in the CNS, has been found to decrease after neurologic insult as a result of axonal injury [14]. It has been further shown that the synthesis of NAA is localized to neuronal mitrochondria [15]. To study the pathogenesis and potential treatment of neurologic sequelae after cardiac surgery, we have developed a canine model of HCA. Using this model, we detected changes in NAA after neurologic injury by MRS and compared these results with fresh tissue NAA extraction and clinical neurologic assessment. In addition, we evaluated the impact of the neuroprotective agent diazoxide on NAA levels.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Preparation
All experiments were preapproved by the Johns Hopkins School of Medicine Animal Care and Use Committee and performed in compliance with the "Guide for the Care and Use of Laboratory Animals," published by the National Research Council (National Academy Press, 1996). Our canine model of HCA has been previously described in the literature [8, 9, 12]. The canines were male, heartworm-free, 6- to 12-month-old mongrels weighing 27 to 30 kg. Animals were anesthetized with sodium pentobarbital (30 mg/kg), endotracheally intubated, and maintained on halothane (0.8% to 2%) inhalational anesthetic through a Narkomed (Draeger Medical Inc, Telford, PA) anesthesia ventilator. Continuous electrocardiographic monitoring was employed. Esophageal, rectal, and bilateral tympanic membrane thermoprobes were placed to monitor temperature throughout the protocol. The left femoral artery was cannulated to monitor arterial pressures and to draw blood for blood gas analysis.

Cardiopulmonary Bypass and Hypothermic Circulatory Arrest
The cardiopulmonary bypass (CPB) circuit consisted of a Cobe membrane oxygenator (Cobe Laboratories, Lakewood, Colorado), a Sarns Roller pump system (Sarns, Ann Arbor, Michigan), and a 40 µm arterial filter. The circuit was primed with lactated Ringer's solution with potassium chloride (10 mEq). After heparinization (300 U/kg intravenously [IV]), the right femoral artery was cannulated, and the cannula (12F to 14F) was advanced into the descending thoracic aorta. Venous cannulae (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 (approximately 30 minutes). Pump flows from 80 to 100 mL · kg^-1 · min^-1 were required to maintain a mean arterial pressure of 50 to 60 mm Hg. The pump was then turned off, and venous blood drained by gravity into the reservoir. Circulatory arrest was maintained for 2 hours, followed by reinstitution of CPB and rewarming (approximately 2 hours). At 36°C the animals were defibrillated as necessary, weaned from CPB, and decannulated. After decannulation, the animals remained on the operating table and were monitored intensively. Mechanical ventilation was maintained until arterial blood gases and ventilatory efforts assured successful extubation. Fentanyl (10 to 20 mg/kg IV) and midazolam (1 mg IV) were used in the early postoperative period. Buprenorphine (0.3 mg IV) was given once a day, and as needed for long-term pain control.

Experimental Design
Canines were divided into three groups (n = 15/group): normal (unoperated), HCA (HCA only), and HCA+diazoxide (pharmacologic treatment before HCA). Animals in the HCA+diazoxide group received a bolus of diazoxide (2 mg/kg) immediately before the initiation of CPB. A diazoxide infusion (0.5 mg/min) was started after the administration of the bolus and was continued for a total of 1 hour, 45 minutes during the cool down and rewarming periods. Animals in the HCA group received vehicle only. All canines underwent magnetic resonance imaging and spectroscopy at 24 hours, immediately followed by sacrifice and brain procurement for tissue analysis.

Clinical Neurologic Assessment
Separate cohorts of HCA (n = 16) and HCA+diazoxide (n = 23) animals were followed for 72 hours after HCA with neurologic assessment performed every 24 hours. The species-specific behavior scale used in this study was validated at the International Resuscitation and Research Center, University of Pittsburgh [16]. Five components of neurologic function were evaluated: level of consciousness; respiratory 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). Two independent observers evaluated each animal daily using this system and the mean score was recorded.

Magnetic Resonance Spectroscopy
All MRS studies were performed at 24 hours after HCA on a 1.5 Tesla Philips Gyroscan-NT MR scanner (Philips Medical Systems, Best, Netherlands). A commercially available C4 type, 8-cm diameter high-spatial-resolution surface coil (Philips) was used for data acquisition. The animal was sedated with pentobarbital sodium (Nembutal), 0.2 to 0.3 mg/kg IV, intubated for airway protection, and placed in the scanner in supine position with the head directly over the surface coil. Continuous oxygen saturation and electrocardiographic monitoring were performed throughout the scan. The MRS protocol consisted of routine brain anatomic imaging, including a sagittal T1-weighted and axial T2-weighted fast spin-echo magnetic resonance imaging scan. Single voxel-proton spectroscopy was then performed separately for each of the three brain regions of interest: superior parietal cortex, cerebellum, and hippocampus. Single voxel-MRS data from the parietal cortex were acquired using point resolved spectroscopy with a TR/TE of 2,000/140 msec, 128 averages, and voxel dimensions measuring 15 mm in each direction. The scan time was approximately 5 minutes. Single voxel-MRS data for the cerebellum and hippocampus were acquired using a Point Result Spectroscopy Sequence (PRESS) sequence with a TR/TE of 2,000/35 msec, 256 averages, and voxel dimensions of 12 mm in each direction. The approximate scan time for each region was 9 minutes. The data were processed using in-house software [17]. Spectra were processed with eddy-current and zero-order phase correction based on the water peak, 3 Hz Gaussian line-broadening, and baseline correction [18]. After assigning the chemical shift of NAA to 2.02 ppm, various brain metabolite peak areas were determined using curve fitting to a Gaussian function over the following frequency ranges: choline (Cho, 3.34 to 3.14 ppm), creatine/phosphocreatine (Cr, 3.14 to 2.94 ppm), and NAA (2.22 to 1.82 ppm). The NAA:Cho, NAA:Cr, and Cho:Cr metabolite ratios were obtained using the peak area values.

NAA Extraction
All animals were sacrificed by exsanguination under full anesthesia, and the brains were perfused with 12 L ice-cold saline at 100 mm Hg through an aortic cannula. The brains were then harvested and sectioned. The tissue was frozen on dry ice and stored at –80°C.

Approximately 40 mg tissue was homogenized in 0.1 M salt-saturated HCl, then extracted into ethylacetate. The ethylacetate was evaporated, and samples were derivatized with N,O,-bis(trimethylsilyl) trifluoroacetamide plus 1% trimethylchloro-silane. The NAA was quantified in a blinded manner by stable isotope dilution gas chromatography/mass spectrometry, using ¹⁵N(²H₃acetyl)-L-aspartic acid as an internal standard [19]. Tissue was sampled separately from the superior parietal cortex, cerebellum, and hippocampus for each animal.

Statistical Analysis
All statistics are reported as mean ± SD. Comparisons between groups were made by Student's t test and analysis of variance (ANOVA). A p value of 0.05 or less was considered significant.

	Results

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Clinical Neurologic Assessment
The Pittsburgh Canine Neurologic Scoring System was employed daily through the 72-hour survival period to assess the clinical neurologic status of each animal. The neurologic injury that results after 2 hours of circulatory arrest renders these animals severely ataxic, with an overall decreased level of consciousness and inability to perform routine daily tasks such as eating, drinking, sitting, or standing. However, canines in the HCA+diazoxide group fared much better than those in the HCA group, with an improved level of consciousness and ability to perform some of these daily tasks. The neurologic scores were 229, 200, and 151 for the HCA group versus 200, 159, and 121 in the HCA+diazoxide group, at 24, 48, and 72 hours, respectively (0 = normal; 500 = brain death). These data are summarized in Figure 1, which demonstrates a statistically significant improvement in the HCA+diazoxide group at all time points.

View larger version (53K): [in this window] [in a new window]   Fig 1. Clinical neurologic scores using the Pittsburgh canine neurologic scoring system (0 = normal; 500 = brain death). (Dark gray bars = hypothermic circulatory arrest [n = 16]; light gray bars = hypothermic circulatory arrest plus diazoxide [n = 23]; hr = hours.)

View larger version (54K): [in this window] [in a new window]   Fig 2. Magnetic resonance spectroscopy–measured N-acetyl-asparate:choline (NAA:Cho) ratios for the cortex, cerebellum, and hippocampus. The p values compare normal animals (black bars; n = 15) versus hypothermic circulatory arrest (HCA) animals (dark gray bars; n = 15); *p values compare HCA versus HCA+diazoxide animals (light gray bars; n = 15). (NS = not significant.)

View larger version (44K): [in this window] [in a new window]   Fig 3. Fresh brain tissue N-acetyl-asparate extraction determined by mass spectrometry for the cortex, cerebellum, and hippocampus. The p values compare normal animals (black bars; n = 15) versus 24-hour hypothermic circulatory arrest (HCA) animals (dark gray bars; n = 15); *p values compare HCA versus 24-hour HCA+diazoxide animals (light gray bars; n = 15).

	Comment

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The neurologic injury that results after 2 hours of circulatory arrest is significant. The animals typically have a depressed level of consciousness and are severely ataxic. This 2-hour time frame was chosen to provide a reproducible injury pattern and also to investigate the most severe injury. Our data show a daily improvement in the neurologic scores of the animals over the 72-hour survival period, representing normal postoperative recovery. However, animals pretreated with diazoxide consistently had improved clinical neurologic scores at each time point versus the HCA group, suggesting a significant neuroprotective effect of this pharmacologic agent. Given the role of diazoxide as a mitochondrial adenosine triphosphate–dependent K⁺ channel agonist, this provides further evidence for mitochondrial energy failure as an important mechanism of neuronal injury.

The Pittsburgh Canine Neurologic Scoring System is a crude measure of the overall neurologic status of the animal that can be very effective in assessing the injury in certain regions of the brain but inadequate for others. For example, this protocol can indirectly measure cerebellar dysfunction through its motor evaluation; however, the hippocampus, which is involved with memory and learning, is much more difficult to assess in an animal. This represents one limitation of our analysis.

To further assess the clinical improvement observed with diazoxide pretreatment, we sought to provide a more objective, clinically useful, postoperative evaluation. Therefore, routine MR imaging was performed at 24 hours after the period of HCA, and included T2- and diffusion-weighted imaging. Despite the significant neurologic impairment of the canines undergoing circulatory arrest without neuroprotection, no obvious abnormalities were identified on imaging. This finding only further substantiated the subtle nature of the injury pattern, which is believed to occur on a subcellular, mitochondrial level [21]. Proton MRS, however, allowed us to detect and quantify subcellular metabolic changes within the brain after HCA. The most significant finding on MRS was a decline in the NAA:Cho ratio after HCA, which is a known marker of neuronal mitochondrial dysfunction. Studies have shown significant reductions in NAA:Cho and NAA:Cr ratios after a traumatic brain injury [20]. Reduced NAA values represent either neuronal loss or dysfunction and are believed to be a result of axonal injury [22].

Magnetic resonance spectroscopy–measured NAA:Cho ratios were significantly decreased in the cortical grey matter of the superior parietal cortex after HCA, reflecting the severe neurologic injury associated with HCA. Pretreatment with diazoxide limited neurologic injury, reflected in a preserved NAA:Cho ratio. These data suggest improved metabolic function in diazoxide-treated canines, which is consistent with the clinical neurologic improvement seen in these animals. It has been previously demonstrated that NAA is synthesized in neuronal mitochondria and its synthesis has also been correlated with adenosine triphosphate production [23]. Similar statistically significant trends were not observed in the cerebellum or hippocampus. This may represent a limitation of the current level of sophistication of MRS. We are currently acquiring spectroscopic data using a 12- to 15-mm voxel that may be too large to get precise readings from a small area of the brain such as the hippocampus. As this technology improves, so will our ability to more accurately quantitate these subcellular metabolites.

Fresh brain tissue NAA extraction was performed by mass spectrometry immediately after the MR imaging and spectroscopy. In all three brain regions measured (cortex, cerebellum, and hippocampus), there were statistically significant decreases in NAA levels after HCA. In addition, the HCA+diazoxide group demonstrated preservation of near-normal NAA levels in all three regions. We believe this biochemical quantification is an important adjunct to substantiate the NAA reduction seen with MRS. It also provides additional evidence of the neuroprotective effect of diazoxide in our canine model of HCA.

Hypothermic circulatory arrest facilitates surgical repair of complex cardiovascular lesions, but it is not without adverse clinical consequences. Neurologic injury remains a significant complication owing to the extreme sensitivity of the central nervous system to ischemia. Much research has focused both on the mechanism of neurologic injury, as well as on potential mitigation of such injury by pharmacologic preconditioning. As newer technologies such as MRS continue to develop, the ability to assess neurologic injury will improve. Our MRS data was consistent with the clinical and histologic findings in this canine model of HCA. We have demonstrated an alteration in the NAA:Cho ratio after HCA using this technology, as well as an improved ratio after diazoxide pretreatment. These findings were further substantiated with biochemical analysis of NAA levels in the CNS. Therefore, we believe that MRS offers an early, noninvasive means of evaluating postsurgical neurologic injury and the effect of various neuroprotective agents.

	Acknowledgments

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This study was supported by the Dana and Albert Broccoli Center for Aortic Diseases, the Mildred and Carmont Blitz Cardiac Research Fund, and the National Institutes of Health (NIH R37[NS31238-10], William A. Baumgartner-PI). Doctor Christopher Barreiro is a Hugh R. Sharp, Jr, Research Fellow, and Drs Jason Williams and Torin Fitton are Irene Piccinini Investigators. Doctor William Baumgartner is the Vincent L. Gott Professor of Cardiac Surgery. The authors would also like to thank Melissa Jones, Jeffrey Brawn, and Tamara Treat for their contribution of outstanding technical assistance. This project could not have been completed without their participation.

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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): Christopher J. Barreiro Jason A. Williams Torin P. Fitton Vincent L. Gott Michael V. Johnston William A. Baumgartner Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Barreiro, C. J. Articles by Baumgartner, W. A. Search for Related Content PubMed PubMed Citation Articles by Barreiro, C. J. Articles by Baumgartner, W. A. Related Collections Cerebral protection