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Ann Thorac Surg 2006;81:2235-2242
© 2006 The Society of Thoracic Surgeons
a Division of Cardiac Surgery, The Johns Hopkins Medical Institutions and the Kennedy-Krieger Institute, Baltimore, Maryland
b Department of Neurology, The Johns Hopkins Medical Institutions and the Kennedy-Krieger Institute, Baltimore, Maryland
c Division of Neuropathology, The Johns Hopkins Medical Institutions and the Kennedy-Krieger Institute, Baltimore, Maryland
Accepted for publication December 20, 2005.
* Address correspondence to Dr Baumgartner, Division of Cardiac Surgery, The Johns Hopkins Hospital, 600 N Wolfe St, Blalock 618, Baltimore, MD 21287; (Email: wbaumgar{at}csurg.jhmi.jhu.edu).
Presented at the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.
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Abstract |
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METHODS: Thirteen dogs pretreated with valproate before 2 hours of hypothermic circulatory arrest survived for 24 hours (n = 7) or 72 hours (n = 6). Thirteen control animals (placebo only) also survived for 24 hours (n = 7) or 72 hours (n = 6) after hypothermic circulatory arrest. Blinded clinical neurologic evaluation was performed daily until sacrifice using the Pittsburgh Canine Neurologic Scoring System. Brains were harvested for blinded histopathologic analysis by a neuropathologist to determine the extent of apoptosis and necrosis in 11 brain regions (Total Brain Cell Death Score: 0 = normal, 99 = extensive neuronal death in all regions). Quantification of N-acetyl-aspartate, an established marker for brain injury, was performed with mass spectrometry.
RESULTS: Valproate dogs scored significantly better than control animals on clinical neurologic evaluation. Histopathologic examination revealed that valproate animals demonstrated less neuronal damage (by Total Brain Cell Death Score) than control animals at both 24 hours (16.4 versus 11.4; p = 0.03) and 72 hours (21.7 versus 17.7; p = 0.07). At 72 hours, the entorhinal cortex, an area involved with learning and memory, was significantly protected in valproate dogs (p < 0.05). Furthermore, the cortex, hippocampus, and cerebellum demonstrated preservation of near-normal N-acetyl-aspartate levels after valproate pretreatment.
CONCLUSIONS: These data demonstrate clinical, histologic, and biochemical improvements in dogs pretreated with valproate before hypothermic circulatory arrest. This commonly used drug may offer a promising new approach to neuroprotection during cardiac surgery.
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Introduction |
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Valproic acid (valproate) is an anticonvulsant that is used in both children and adults, and has proven to be one of the safest and best tolerated antiepileptic drugs available [4]. It is also commonly used in the treatment of bipolar disorder, neuropathic pain, and migraines. On the basis of the diverse mechanisms of action of this drug, recent interest has focused on using valproate as a neuroprotectant for neurodegenerative diseases. The most likely means by which valproate protects the brain against ischemia seems to lie in its inhibition of histone deacetylase. Both in vitro and in vivo models using valproate for neuroprotection have demonstrated increased amounts of acetylated histone H3, accompanied by decreased cell damage and improved clinical outcomes [5, 6].
On the basis of these promising results using valproate for cerebral protection after ischemic insults, our group applied this agent to our canine model of HCA. Previous work with our canine preparation demonstrated that it provides a clinically relevant model that enables testing of therapeutic interventions in a controlled, reproducible environment [7–12]. Our aim was to demonstrate clinical neurologic improvements as assessed using a dog-specific behavior scale that has been validated at the International Resuscitation and Research Center, University of Pittsburgh [13]. In addition, we hypothesized that the clinical improvements seen in animals treated with valproate before HCA should reflect neuronal protection, as demonstrated by histologic evaluation, and preservation of N-acetyl-aspartate (NAA) levels, a well-known marker of brain injury [14, 15].
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Material and Methods |
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Cardiopulmonary Bypass and Hypothermic Circulatory Arrest
The cardiopulmonary bypass (CPB) circuit consisted of a Cobe membrane oxygenator (Cobe Laboratories, Inc, Lakewood, CO), a Sarns Roller pump system (Sarns, Inc, Ann Arbor, MI), and a 40-µm arterial filter. The circuit was primed with lactated Ringer's solution with potassium chloride (10 mEq). After heparinization (300 U/kg intravenously), the right femoral artery was cannulated (12F to 14F), and the cannula was advanced into the descending thoracic aorta. Venous cannulas(18F to 20F) were advanced to the right atrium from the right femoral and external 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 and weaned from CPB, and the cannulas were removed. After removal of cannulas, the animals remained on the operating table and were monitored for recovery. Mechanical ventilation was maintained until arterial blood gases and ventilatory efforts assured successful extubation. Fentanyl (10 to 20 mg/kg intravenously) and midazolam (1 mg intravenously) were used in the early postoperative period. Buprenorphine (0.3 mg intravenously) was given once a day, and as needed for long-term pain control.
Experimental Design
Animals were divided into four groups based on treatment before HCA and length of survival. Thirteen dogs were treated with a 750-mg bolus (25 mg/kg) of valproic acid (sodium valproate, DepaconTM) before the institution of CPB. This was followed by a continuous infusion of sodium valproate, 2.25 g (75 mg/kg), during the course of 2 hours 30 minutes during cool-down and rewarming while on CPB. The infusion was stopped during HCA. Seven dogs in the valproate group survived for 24 hours, and 6 dogs survived for 72 hours after HCA. Thirteen control animals received only placebo (0.9% normal saline solution) in the same volume as the valproate bolus and infusion. Control animals also survived for 24 hours (n = 7) or 72 hours (n = 6) after HCA. Investigators involved in the clinical, histologic, and biochemical evaluation of these animals remained blinded to the treatment group until the completion of the study.
Clinical Neurologic Assessment
Clinical neurologic assessment was performed on all animals at 24-hour intervals until sacrifice. The dog-specific behavior scale used in this study was validated at the International Resuscitation and Research Center, University of Pittsburgh [13]. There were five components of neurologic function evaluated: level of consciousness, 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 possible score of 0 (normal) to 500 (brain death). Two independent observers evaluated each animal daily using this system, and the mean score was recorded.
Histopathology
After being observed clinically for 24 or 72 hours, all animals were euthanized by exsanguination under full anesthesia. In preparation for euthanasia, animals underwent median sternotomy and cannulation of the ascending aorta using a 22F cannula. Cardiopulmonary bypass was initiated after clamping the descending aorta to ensure the brains were perfused with 12 L of ice-cold saline solution (4°C) at 100 mm Hg. The right atrial appendage was transected, and the venous return was suctioned into a reservoir. After perfusion, the brains were harvested, and the right hemibrain was fixed in 10% formalin and embedded in paraffin. Paraffin embedded tissues were then sectioned into 8-µm slices and were stained with hematoxylin and eosin stains for histologic evaluation. The left hemibrain was sectioned, frozen with dry ice, and stored at –80°C for future biochemistry studies.
Sections were examined by light microscopy for neuronal apoptosis or necrosis in a blinded manner by a neuropathologist (J.C.T.). Criteria for apoptosis included cellular shrinkage and condensation of nuclear chromatin into sharply delineated and regularly shaped round or crescent masses. Necrotic neurons were identified by shrinkage of the perikaryon, effacement of the Nissl bodies, intense eosinophilic staining of the cytoplasm, condensation of the nuclear chromatin, and nuclear pyknosis. Some necrotic neurons displayed karyorrhexis characterized by dissolution of nuclear chromatin into small and irregular aggregates.
Eleven different regions of the brain were examined for the extent of apoptosis and necrosis using hematoxylin and eosin staining: midfrontal cortex; superior parietal cortex; caudate; hippocampus—dentate gyrus; hippocampus—pyramidal gyrus; entorhinal cortex; amygdala; cerebellum—molecular layer; cerebellum—Purkinje layer; cerebellum—granule layer; and brainstem. Each region received separate scores for apoptosis and necrosis (Table 1) according to the worst scoring high-powered field in the region. Once all regions had been scored, the apoptosis and necrosis scores for each region were added to determine a Total Brain Cell Death Score (0 = normal, 99 = extensive neuronal death in all regions), which provided a semiquantitative measurement that was used for statistical comparison among the groups.
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Statistical Analysis
Statistical analysis was performed using GraphPad Software (GraphPad Software, Inc, Del Mar, CA). Comparisons among groups were made using a two-tailed Student's t test and analysis of variance. All statistics are reported as mean ± standard error of the mean unless otherwise indicated. A p value less than or equal to 0.05 was considered significant.
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Results |
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Clinical Neurologic Assessment
Animals treated with valproate demonstrated significantly better clinical neurologic function at all time points after HCA (Fig 1). Mean neurologic scores for valproate animals at 24, 48, and 72 hours after HCA was 165.6 ± 9.9, 121.1 ± 9.9, and 97.2 ± 5.2, respectively. These scores were significantly better than control animals at each time point, which demonstrated mean scores of 196.8 ± 8.2, 190.5 ± 15.0, and 137.2 ± 6.4, respectively (p = 0.02; p < 0.001; p < 0.001).
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Histopathology
Valproate animals demonstrated less neuronal damage than controls on histopathologic examination. Animals sacrificed 24 hours after HCA demonstrated a Total Brain Cell Death Score of 11.4 ± 3.7, which was significantly better than the Total Brain Cell Death Score of 16.4 ± 3.7 for control animals at the same time interval (p = 0.03). Similar trends were observed for valproate versus control dogs at 72 hours after HCA (17.7 ± 1.5 versus 21.7 ± 4.6, p = 0.07).
Subset analysis of individual regions also revealed that certain areas were significantly more protected after treatment with valproate. At 24 hours after HCA, the superior parietal cortex (0.9 ± 0.7 versus 1.6 ± 0.5, p = 0.05), amygdala (0.3 ± 0.5 versus 2.3 ± 1.7, p = 0.01), and granule layer of the cerebellum (0.1 ± 0.4 versus 0.7 ± 0.5, p = 0.03) each demonstrated a significant protective effect in dogs receiving valproate. Furthermore, dogs surviving for 72 hours demonstrated significant protection from apoptotic cell death in the entorhinal cortex (0 versus 0.5 ± 0.5, p < 0.05), an area critical for learning and memory. Finally, the molecular layer of the cerebellum in the 72-hour-survival valproate animals exhibited significant protection from necrosis (0.5 ± 0.5 versus 2.5 ± 1.6, p = 0.02), which resulted in significant protection in the cerebellum overall in these animals (1.0 ± 0.2 versus 2.4 ± 1.5, p = 0.05). Trends toward histologic protection were also seen in the dentate gyrus of the hippocampus in 24-hour-survival animals (p = 0.07), as well as the granule layer of the cerebellum (p = 0.09) and the superior parietal cortex in 72-hour-survival animals (p = 0.17).
N-Acetyl-Aspartate Extraction Using Mass Spectrometry
Mass spectrometry was used to quantitate the amount of NAA present in the brain tissue of dogs that underwent HCA. Table 2
indicates the significant preservation of NAA levels in dogs treated with valproate before HCA versus control animals that did not receive pretreatment. N-acetyl-aspartate levels of valproate and control animals are compared with a cohort of normal dogs (n = 7) that did not undergo HCA, but that were sacrificed using the same cold saline infusion technique. Analysis of variance was performed to determine statistical differences among the groups.
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Comment |
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Two of the most promising mechanisms, however, involve the prevention of glutamate excitotoxicity and the inhibition of histone deacetylation. Cumulative previous work in our laboratory has demonstrated that glutamate excitotoxicity is primarily responsible for the neurologic damage sustained in dogs undergoing prolonged HCA [7, 8]. Valproate has been shown to reduce the extracellular accumulation of glutamate in a rat model of malonate toxicity, which paralleled the pathologic findings of reduced cortical lesions in treatment animals versus controls [18]. In this and other studies performed on rat cerebral cortex, valproate was also found to significantly increase levels of heat shock protein 70 and inhibit histone deacetylation [5, 6, 18].
Heat shock proteins are well known for their cytoprotective effects. However, the effects of histone acetylation and hyperacetylation are currently under investigation as potentially significant mechanisms for cytoprotection. Deacetylated histones are produced when histone deacetylase enzymes catalyze the hydrolysis of acetyl groups on certain amino acids in the histone proteins. Deacetylated histones bind DNA with high affinity, leading to chromatin condensation and reduction in transcription factor activity [19]. Histone deacetylase inhibitors, such as valproate, have been shown to cause acetylation or hyperacetylation of histones, which release from DNA and allow the transcription of enzymes that protect against oxidative stress [19, 20].
We chose a circulatory arrest time of 2 hours for our canine model of HCA because of the need for an exaggerated injury response in these animals. Previous experience in our laboratory with shorter HCA durations led to no definable clinical injury after surgery. To asses the effects of different neuroprotective strategies, our model needed to be severe enough to induce a significant injury in these dogs. Our experience has shown that 2 hours provides this extreme injury without causing fatal neurologic sequelae in these study subjects.
The present study using our canine model of HCA validates the claims that valproate acts as a significant neuroprotectant during episodes of cerebral ischemia. We chose a total dose of 3 g divided into a 750-mg bolus and a 2.25-g infusion during 2 hours 30 minutes from preliminary dosing studies. Clinically relevant serum levels of valproate are reported to be in the range of 30 to 100 µg/mL, although the drug appears to be most efficacious at levels greater than 50 µg/mL [21]. Dosing studies performed on the first dog in this series and on a dog not undergoing HCA revealed that our dosing regimen enabled these animals to achieve serum levels greater than 50 µg/mL before HCA and for 6 hours after cerebral insult (data not shown). Furthermore, cerebrospinal fluid studies on a dog not undergoing HCA revealed that valproate did penetrate the blood–brain barrier in these dogs (data not shown). Therefore, the clinical, histologic, and biochemical improvements seen in these experiments can be attributed to clinically relevant dosing of this drug.
Clinical neurologic assessment provides the most tangible evidence for neuroprotection in the current study. Our neurologic scoring protocol has been validated in multiple previous studies, both by our group and others [7–13]. The system is subjective, based on the individual evaluator's assessment of each individual dog's function; however, we attempted to minimize the effects of this subjectivity by having two blinded, independent observers evaluate each animal.
Although both valproate animals and control animals exhibit clinical evidence of significant neurologic impairment, valproate animals demonstrated better preservation of higher cortical function and motor and sensory function when compared with control animals. Furthermore, valproate animals improved more quickly with time when compared with control animals, indicating the beneficial effects this drug may have on both cerebral protection and cerebral recovery after neurologic insults.
The histopathology findings in this study support the conclusions drawn from clinical neurologic assessment. By combining the necrosis and apoptosis scores of each region into a single Total Brain Cell Death Score, we are able to highlight the global neurologic injury sustained by these animals. The fact that animals receiving valproate demonstrate less overall histopathologic injury at both 24 and 72 hours after HCA provides a reasonable explanation for the clinical findings of improved level of consciousness and motor function, as well as the trends in improved behavior, respiratory patterns, and cranial nerve function.
Additionally, we were still able to demonstrate significant improvements in certain key regions of the brain, such as the entorhinal cortex, superior parietal cortex, amygdala, and cerebellum. Each of these regions are involved in higher cortical functions, such as learning and memory, or motor and sensory functions, which correlate well with the clinical neurologic findings of improved level of consciousness, motor function, and sensory function in the valproate animals. However, the fact that some regions of the brain only exhibited trends toward histopathologic improvement underscores the importance of finding better markers for neurologic injury so that interventions to prevent these injuries can be assessed more effectively.
N-acetyl-aspartate is a ubiquitous molecule in the central nervous system, and is the most abundant amino acid in the brain. Although the function of NAA remains controversial, research has determined that NAA levels decrease after neurologic insult [14, 22]. This reduction in NAA represents neuronal loss or dysfunction, presumably as a result of axonal injury to these neurons [23]. Measuring NAA levels in the brains of dogs undergoing HCA is another well-documented method for evaluating the neurologic damage sustained by these animals and the neuroprotection provided by valproate [14, 15]. The fact that valproate animals demonstrated significant preservation of near-normal NAA levels at both 24 and 72 hours after HCA provides further evidence to substantiate the use of valproate as a neuroprotectant during ischemia.
Our study is limited, in part, by the subjectivity of the clinical scoring system, although we attempted to reduce the effects of this subjectivity as much as possible by using two independent reviewers. Furthermore, although the scoring system does a good job of evaluating certain brain functions, it can be inadequate for assessing others. For example, this protocol can indirectly measure cerebellar dysfunction through its motor evaluation; however, the hippocampus, which is involved in learning and memory, is much more difficult to assess in an animal. However, the improvements seen on histology and biochemical analysis using NAA validate the findings seen on clinical neurologic assessment with objective data.
To validate the findings of this study, the mechanisms of actions of valproate in this model will need to be verified. Current possible methods for achieving this include using the newly described canine genome, which can allow us to analyze and identify the gene transcriptional changes that occur during HCA and during treatment with valproate. By identifying the genetic changes that occur in this injury model, we will be able to target our therapy toward specific, relevant targets. Valproate will hopefully provide a springboard for such future studies.
We believe that the results reported in this study offer a simple, clinically available method for providing neuroprotection to patients undergoing cardiac surgery that can potentially improve long-term outcomes. Valproate has the advantages of clinical availability, time-tested clinical safety, and multiple mechanisms of cytoprotection. As these mechanisms become better understood, valproate may be a drug that makes a simple, effective transition from laboratory studies to clinical protocols. The clinical, histologic, and biochemical improvements seen in our dogs treated with valproate before HCA indicate that this drug may offer a promising new approach to neuroprotection during cardiac surgery.
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Discussion |
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DR WILLIAMS: Thank you, Dr Hammon, and thank you for your kind comments, especially in light of your immense contributions to the field of neuroprotection during cardiac surgery. Prior to initiating this study, we performed dosing experiments on animals that had not undergone HCA. We know that the therapeutic range for this drug is fairly wide, but the accepted therapeutic serum level for valproic acid in humans is 50 mg/dL, so this was our target level. We performed dosing studies in animals that did not undergo HCA in order to determine what dose would be appropriate for obtaining those levels. We found that dogs actually metabolize valproic acid much quicker than humans. So, we had to use much larger doses of valproic acid in these dogs than we would normally use in humans to achieve the same therapeutic level. In addition, the dogs' rapid metabolism of this drug is actually why we used an infusion instead of a simple IV bolus. The combination of the bolus plus infusion enabled these animals to maintain therapeutic levels over the course of six hours during the experiment. (Slide) This slide demonstrates representative serum valproate levels taken from a dog that actually underwent HCA. You can see that over the course of six hours following the initial bolus of drug, the animals maintained a therapeutic level of valproate. We obtained these samples simply to validate that the therapeutic levels would stay the same in HCA dogs. So the answer to your question is the therapeutic levels we achieved are relevant to humans but the actual doses that we gave to these dogs are higher than we would have to give to a human being. The second part of that question deals with the toxicity of this drug at the doses we used. In humans, the large doses we had to give these animals may have some toxicity. However, as I just mentioned, we would not need to give humans the same dose that we gave these dogs. At normal human doses, where we could achieve a therapeutic serum level, we would not anticipate any significant toxicity. Your second question refers to the NAA levels in the cerebellum of these animals. We certainly agree that in our model, the cerebellum has exhibited significant damage in the past, and protection of the cerebellum has been an area of intense interest to us. Ironically, these dogs actually exhibited fairly good motor function following treatment with valproate, although we would assume that ataxia would be a problem because of the cerebellar damage that we see histologically. In fact, these dogs actually do not have the ability to sit, stand, or walk following this injury, and some of this may be due to the cerebellar dysfunction that occurs. But these animals did have good recovery of other motor functions, which is a bit of a paradox based on the amount of histological damage we see in the cerebella of these animals. Finally, regarding our plans for the future, we always have to be careful to extrapolate animal data to humans. However, because this drug is FDA approved, the safety profile is relatively good and many physicians have excellent experience with it. Based on our results, we feel that it is worth further investigation in humans. We are in the early stages of seeking institutional review board approval for a pilot study. Our goal for this study will be to do a safety profile in a small cohort of patients that are undergoing these types of procedures. Once we have those data, we can move to the next phase of human investigation, which would be a randomized control trial.
DR GRAYSON H. WHEATLEY III (Phoenix, AZ): I want to compliment you on a nice study and excellent presentation. I had one question about your study design. I saw that there was a negative control in that seven animals were sacrificed that did not receive hypothermic circulatory arrest nor valproic acid, but I didn't see a positive control, ie, animals that did not undergo hypothermic circulatory arrest and recevied valproic acid, and whether or not you think there might be some relevance there when comparing your NAA levels?
DR WILLIAMS: Thank you, Dr Wheatley. That is an interesting point and one that is certainly worth investigating. We did not do that as part of this study partly because we are trying to be judicious with the amount of animals that we use in this model. But that is certainly an interesting and noteworthy critique. It would be beneficial to determine whether or not valproate inherently increases the NAA levels in these animals. Unfortunately, we don't have those data.
DR NICHOLAS T. KOUCHOUKOS (St. Louis, MO): This is an excellent experimental study. There is, in the clinical setting however, an increasing tendency in both the adult and pediatric populations, to reduce the intervals of circulatory arrest to levels of 30 and even 15 minutes or less to mimimize the neurologic insult. I know that the reason for using two hours of circulatory arrest in the laboratory is to produce a severe ischemic insult. But isn't it important, based upon what is happening clinically, to start looking at what we can do with ischemic insults of 15 and 30 minutes in terms of adjunctive pharmacologic support? I would encourage you to look at lesser intervals of circulatory arrest to see if these drugs are effective adjuncts. Based upon your studies, would you be comfortable recommending the use of this drug in a clinical situation where the circulatory arrest period would not exceed 15 to 30 minutes?
DR WILLIAMS: Thank you for those comments and questions, Dr Kouchoukos. I think you make a valid point that clinically we would be using this drug in patients that undergo circulatory arrest for a much shorter amount of time. The problem with our model is that arrest times less than an hour and a half to an hour and 45 minutes do not really produce much in the way of clinical findings. Therefore, if we arrest dogs for only 30 to 60 minutes, it is very difficult to compare control and treatment animals because the control animals do not exhibit any clinical findings. It is simply a limitation of this model that we are not able to shorten the circulatory arrest time. Because we haven't even performed the pilot studies for safety profiles in this patient population, I don't think we are comfortable recommending using this drug in any patient population yet. But I think those studies soon will be forthcoming, and at that point we will be able to make a better assessment.
DR PAUL KURLANSKY (Miami, FL): What you presented is some very compelling evidence about reperfusion injury in the cerebral circuit. The question that I have is whether or not you had thought of applying this in two other areas: one was in the stroke population and the other is in the area of reperfusion injury to the myocardium. Is there any evidence regarding valproic acid and its impact on myocardial cells and whether it could in fact be effective in ischemic reperfusion issues for the myocardium as well as for the brain?
DR WILLIAMS: Thank you for your questions, Dr Kurlansky. I think those are two very interesting points and I appreciate those comments. In fact, this field of research using valproic acid for cerebral protection actually began with rodent models of middle cerebral artery occlusion. So this concept actually began with stroke models, and this is an area of research that is still ongoing. With regards to the myocardial cells and myocardial protection, it is an interesting idea, and one that we haven't specifically addressed. But this is an idea that certainly could be addressed in the future.
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