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Ann Thorac Surg 2002;73:163-172
© 2002 The Society of Thoracic Surgeons
a Department of Surgery, University of Oulu, Oulu, Finland
b Department of Forensic Medicine, University of Oulu, Oulu, Finland
c Department of Anesthesiology, University of Oulu, Oulu, Finland
d Department of Laboratory of Clinical Neurophysiology, University of Oulu, Oulu, Finland
Accepted for publication August 17, 2001.
* Address reprint requests to Dr Juvonen, Department of Surgery, University of Oulu, PO Box 22 90221 Oulu, Finland
e-mail: tatu.juvonen{at}oulu.fi
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Abstract |
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Methods. Twenty-four pigs undergoing 75-minute period of HCA at 20°C were randomly assigned to receive saline; lamotrigine (20 mg/kg) before HCA (L); or lamotrigine (20 mg/kg) before HCA plus leukocyte filtration before and after HCA (L + LF).
Results. Seven animals (87%) in the L + LF group, 4 (50%) in the L group, and 3 (37%) in the control group were alive on the seventh postoperative day. The median electroencephalogram burst recovery was 94% in the L + LF group (p = 0.024 versus control group), 81% in the L group, and 64% in the control group. Among the surviving animals, the median behavioral scores were 9, 9, and 6 at the seventh day, respectively (p = 0.005 between the L + LF group and the control group). The median histopathologic score was 14 in the L + LF group (p = 0.046 versus control group), 14.5 in the L group (p = 0.062 versus control group), and 21 in the control group.
Conclusions. Lamotrigine has neuroprotective effect during HCA. The combined use of lamotrigine and LF may further improve the survival outcome.
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Introduction |
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Because the development of ischemic brain injury involves several different mechanisms leading to cell damage, targeting a single event may be insufficient to provide effective neuroprotection. We have recently shown in two experimental studies using a chronic porcine model of prolonged HCA that the Na+ channel blocker lamotrigine and leukocyte filtration have neuroprotective effects in this condition [5, 6]. To evaluate whether the combination of these two strategies acting on different phases of ischemic brain injury may improve brain protection during hypothermic global ischemia, we performed the present study in which pigs undergoing 75 minutes of HCA were randomized to receive saline, lamotrigine before HCA, or lamotrigine before HCA plus leukocyte filtration before and after HCA.
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Material and methods |
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Preoperative management
All animals received humane care in accordance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985). The study was approved by the Research Animal Care and Use Committee of the University of Oulu.
Drug administration
An isethionate (2-hydroxyethanesulfonate) salt of lamotrigine [3,5-diamino-6(2,3-dichlorophenyl)-1,2,4-triazine] was diluted in saline to obtain a solution containing lamotrigine at 50 mg/mL and this was packed in 10-mL ampoules in the Pharmaceutical Laboratory of our Institution. Saline placebo ampoules were prepared similarly. A dose of 20 mg/kg was measured and diluted to 50 mL in saline. This volume was given intravenously over a period of 20 minutes, starting 2 hours before HCA.
Anesthesia and hemodynamic monitoring
Anesthesia was induced with medetomidine hydrochloride (0.4 mg/kg intramuscularly), and muscular paralysis was maintained with pancuronium bromide (0.1 mg/kg intravenously). After endotracheal intubation, the animals were maintained on positive pressure ventilation with 35% oxygen. Anesthesia was maintained with isoflurane (1.1% to 1.2%). The arterial catheter was positioned in the left femoral artery. A thermodilution catheter (CritiCath, 7F, Ohmeda GmbH, Erlangen, Germany) was placed through the femoral vein to allow blood sampling, pressure monitoring in the pulmonary artery, and recording of cardiac output. The intracranial temperature probe was placed through a drill hole in the epidural space. The drill hole was positioned 1 cm to the left side from the sagittal joint above a parietal line. Other temperature probes were placed in the esophagus and rectum, and a 10F catheter was placed in the urinary bladder to monitor urine output.
Electroencephalography monitoring
Cortical electrical activity was registered from four stainless steel screw electrodes (5 mm in diameter) implanted in the skull over the parietal and frontal areas of the cortex using a digital electroencephalographic (EEG) recorder (Nervus, Reykjavik, Iceland) and an amplifier (Magnus EEG 32/8, Reykjavik, Iceland). Sampling frequency was 1,024 Hz, bandwidth 0.03 to 256 Hz. All EEG recordings are referenced to a frontal screw electrode, which, together with a ground screw electrode, was implanted over the frontal sinuses. Isoflurane level was adjusted so that the EEG showed steady burst suppression pattern. Then isoflurane end tidal concentration was kept at this steady level until the end of monitoring. The EEG was recorded for 10 minutes to obtain a baseline recording of steady burst suppression activity before the cooling period. After HCA, the EEG recording was restarted and continued until the first postoperative day. The duration of EEG was measured from 5-minute EEG samples at fixed time points, first at 30-minute intervals, and later on at 1-hour intervals. From each 5-minute sample, artifact periods were excluded and from the rest the sum of bursts was counted as percentage of the sum of artifact-free bursts and suppressions. This percentage was used as a measure of EEG activity in the analysis.
Cardiopulmonary bypass
Through a right thoracotomy in the fourth intercostal space, the right mammary artery was ligated, and the heart and great vessels were exposed. A membrane oxygenator (Midiflow D 705, Dideco, Mirandola, Italy) was primed with 1 L of Ringer acetate and heparin (5,000 IU). After heparinization (300 IU/kg), the ascending aorta was cannulated with a 16F arterial cannula, and the right atrial appendage with a single 24F atrial cannula. Nonpulsatile cardiopulmonary bypass (CPB) was initiated at a flow rate of 100 mL/kg per minute and the flow was adjusted to maintain a perfusion pressure of 50 mm Hg. A 12F intracardial sump cannula was positioned in the left ventricle for decompression of the left side of the heart during CPB. In a randomly assigned group, a leukocyte-depleting filter (Leukoguard LG6, Pall Biomedical, Portsmouth, UK) was used throughout CPB before and after HCA. A heat exchanger was used for core cooling. The pH was maintained using the 
-stat principles at 7.40 ± 0.05 with an arterial CO2 tension of 4.0 to 5.0 kPa, uncorrected for temperature. All measurements were performed at 37°C.
A cooling period of 60 minutes was carried out to attain a rectal temperature of 20°C. The ascending aorta was cross-clamped just distal to the aortic cannula. Cardiac arrest was induced by injecting potassium chloride (1 mEq/kg) through the aortic cannula, and topical cardiac cooling was then begun and maintained throughout the aortic cross-clamp period.
Experimental protocol
After cooling to 20°C rectal temperature and cross-clamping the aorta, the animals underwent a 75-minute interval of HCA with the head packed in ice. After this 75-minute period of HCA, rewarming was begun. The left ventricular vent cannula was removed. Weaning from CPB occurred approximately 60 minutes after the start of rewarming with administration of furosemide (40 mg), mannitol (15.0 g), methylprednisolone (80 mg), and lidocaine (40 to 150 mg). Cardiac support was provided by dopamine. The animals were kept in isoflurane anesthesia until the after morning, and then extubated and moved to a recovery room.
During the experiments hemodynamic and metabolic measurements were recorded at five time intervals as follows: at baseline; at the end of cooling (at 20°C, immediately before institution of HCA); during rewarming (at 30°C); 2 hours after the start of rewarming; and 4 hours after the start of rewarming.
Postoperative evaluation
Postoperatively, all the animals were evaluated daily by an experienced observer (V.A.) who was blinded to the study group and who used a species-specific quantitative behavioral score as reported earlier [7]. The assessment quantified the mental status (0 = comatose, 1 = stuporous, 2 = depressed, 3 = normal); appetite (0 = refuses liquids, 1 = refuses solids, 2 = decreased, 3 = normal); and motor function (0 = unable to stand, 1 = unable to walk, 2 = unsteady gait, 3 = normal). Numerical summing of the score of these functions provided a final score. The maximum score of 9 reflected apparently normal neurologic function, whereas lower values indicated substantial brain damage. Animals that died postoperatively were assigned a score "0." Each surviving animal was electively sacrificed on the seventh postoperative day. The entire brain was immediately harvested and weighed, and prepared for subsequent histologic analysis.
Histopathologic analysis
During autopsy, the brain was excised immediately and was immersed in 10% neutral formalin and allowed to fix for 1 to 2 weeks en bloc. Thereafter, 3-mm thick coronal specimens were sliced from the frontal lobe, thalamus (including the adjacent cortex) and hippocampus (including the adjacent brainstem, and temporal cortex), and sagittal specimens from the posterior brainstem (medulla oblongata and pons) and cerebellum. The specimens were fixed in fresh formalin for another week. After fixation, they were processed as follows: rinsing in water for 20 minutes, immersion in 70% ethanol for 2 hours, in 94% ethanol for 4 hours, and 9 hours in absolute ethanol. Thereafter, the specimens were kept 1 hour in an absolute ethanol-xylene mixture, 4 hours in xylene, and then were embedded in warm paraffin for 6 hours. The specimens were then sectioned at 6 µm and stained with hematoxylin and eosin. The sections of the brain specimens of each animal were examined by an experienced senior pathologist (J.H.) who was unaware of the experimental design, and the identity and fate of each animal. The histopathologic signs of injury were scored as follows: 1 = slight edema, dark or eosinophilic neurones or cerebellar Purkinje-cells; 2 = moderate edema, at least two hemorrhages in the section; 3 = severe edema, infarctive foci (local necrosis). A total histologic score was calculated by adding all the regional (cortex, thalamus, hippocampus, posterior brainstem, and brainstem) scores to allow semiquantitative comparisons between the animals.
Microdialysis
The microdialysis catheter (CMA 70; CMA Microdialysis, Stockholm, Sweden) was placed into the brain cortex. A drill hole was positioned 1 cm to the right side of a sagittal joint above a parietal line. The shaft was introduced freehand through a bolt into a depth of 15 mm below the dura. The microdialysis catheter was connected to a 2.5-mL syringe placed in a microinfusion pump (CMA 107; CMA Microdialysis) and perfused with a Ringer solution (Perfusion Fluid; CMA Microdialysis). Samples were collected every 30 minutes. The concentrations of glucose, lactate, glutamate, and glycerol were measured immediately after collection using ordinary enzymatic methods on a microdialysis analyzer (CMA 600; CMA Microdialysis).
Other measurements
Systemic arterial and venous blood samples were obtained to determine pH, oxygen tension, carbon dioxide tension, oxygen saturation, oxygen content, hematocrit, hemoglobin, and glucose levels (Ciba-Corning 288 Blood Gas System, Ciba-Corning Diagnostic Corp, Medfield, MA). Venous lactate levels were measured by a YSI 1500 analyzer (Yellow Springs Instrument Co, Yellow Springs, OH). Leukocyte count was measured by using the Cell-Dyn analyzer (Abbot, Santa Clara, CA).
Statistical analysis
Summary statistics for continuous or ordinal variables are expressed as the median with interquartile range (25th and 75th percentiles) or means with standard deviation (SD). In figures values are shown as medians with interquartile range. The analysis was performed by analysis of variance for repeated measurements. Comparison between relevant time points and baseline (reference category) was performed by paired sample t test or Wilcoxon matched pairs signed rank test. Differences between groups were determined by t test, Mann-Whitney U test, and the Kruskal-Wallis test. The two-tailed Fisher’s exact test was used to evaluate any difference in mortality rates between the study groups. Analyses were performed using a standard commercially available statistical program (SPSS v. 9.0, SPSS Inc, Chicago, IL).
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Results |
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Metabolic data
The concentration of venous lactate increased during cooling and especially after HCA in all groups and returned to baseline levels 4 hours after the start of rewarming (Table 2).
Oxygen consumption and extraction decreased in all groups during cooling. Oxygen consumption decreased in the control group at the 30°C interval and oxygen extraction decreased in the L + LF group 2 hours after the start of rewarming. The differences between the groups were not statistically significant.
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Behavioral outcome
The postoperative behavioral scores of each study group are shown in Figure 3.
Among the surviving animals, the median behavioral scores were 9, 9, and 6 at the seventh day, respectively (p = 0.005 between the L + LF group and the control group).
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Comment |
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The most extensively studied drugs are the glutamate-receptor blockers and Ca2+- and Na+-channel antagonists. The prerequisite for any of these new therapies is their clinical tolerability [9]. Therefore, only a few potentially neuroprotective pharmacologic agents can be considered suitable for clinical use. Lamotrigine, used in clinical practice as an antiepileptic drug, is one of the most intriguing neuroprotective agents. This drug does not seem to have major adverse effects on cardiovascular function and hepatic metabolism and is well tolerated, skin rash being its major adverse event especially in children. It has been shown that lamotrigine improves brain protection after brain ischemia [5, 10–12].
Much attention has been paid also to some of the low-affinity uncompetitive N-methyl-D-aspartate receptor antagonists such as memantine, but this clinically well-tolerated drug failed to show any neuroprotective effect in our surviving pig model [13].
Several studies have shown that during the reperfusion period the interaction between activated leukocytes and capillary endothelium leads to disruption of endothelial integrity and their infiltration into injured tissues, where they cause further tissue injury by altering vasomotor function, generating oxygen free radicals, and releasing cytotoxic enzymes, cytokines, and chemoattractants [4]. We have shown recently that depleting the activated neutrophils by the use of a leukocyte filter can mitigate brain injury during experimental HCA [6]. However, this beneficial effect seems to be related not to a decrease in leukocyte count, but rather to a depletion of activated neutrophils [14]. Such promising results in improving brain protection by targeting different phases of ischemic injury have raised the question whether better brain protection could be achieved by combining several strategies as tested in the present study.
The results of this study confirm the efficacy of lamotrigine as a neuroprotective agent during experimental HCA [5] and suggest that leukocyte filtration when used in combination with lamotrigine may further improve the outcome of animals undergoing 75 minutes of HCA. The benefits of this combined strategy have been described herein in terms of microdialysis findings, EEG recovery, behavioral outcome, histopathologic scores, and survival outcome.
Microdialysis findings were similar in the L + LF and in the L groups, whereas control animals had higher glutamate, glycerol, and lactate levels suggesting the beneficial neuroprotective effects of both strategies. The EEG burst recovery was higher in the L + LF group, a statistically significant difference being found 7 hours after the start of rewarming. As shown in Figure 2, the L group experienced an EEG recovery that was intermediate between the EEG recovery of the L + LF group and the control group. In our previous study using leukocyte filtration, we did not detect any differences in EEG recovery between the leukocyte filtration group and the control group [6], but otherwise a marked difference in EEG burst ratio was observed 4.5 hours after the start of rewarming when lamotrigine was used as compared with controls [5]. Such EEG findings further suggest the potential advantage of a combination therapeutic strategy such as leukocyte filtration plus lamotrigine in this setting.
These findings were coupled with the behavioral outcome scores, the L + LF group achieving significantly better results than controls. Although all the animals of the L group achieved better behavioral recovery than the control group, such a difference failed to achieve a statistical significance probably because few animals survived until the seventh postoperative day.
The histopathologic findings showed a statistically significant better outcome in the animals of the L + LF group as compared with the control group, whereas the difference between the histopathologic scores of the L group and of the control group, despite a trend for a better outcome in the L group, was not statistically significant. The data summarized in Table 4 and Figure 4 seem to suggest no major difference between the L + LF group and the L group in terms of histopathologic scores. However, these two groups differed markedly in terms of survival outcome, which might have masked the detection of more histologically relevant signs of brain injury in pigs that did not survive until the seventh postoperative day.
The combination of leukocyte filtration and lamotrigine achieved satisfactory results also in terms of survival outcome, whereas the use of lamotrigine alone failed to show any significant advantage in reducing the postoperative mortality rate. This observation suggests that, despite the major contribution offered by any neuroprotective strategy, several factors contribute to postoperative mortality after HCA. In fact, leukocyte filtration has been shown to attenuate reperfusion injury in all organs, especially providing effective protection of the heart and lung [15–18]. Therefore, this beneficial effect would add further advantage to any drug with selective brain protective activity such as lamotrigine.
The mortality rates observed in this experimental model could raise some concerns about the value of these findings and of the model itself. However, this chronic experimental model was developed to address major issues in cerebral protection during HCA and to test adjuvant methods. For this reason, we induced extreme conditions that placed the animal at high risk of severe brain injury and death. In fact, a period of 75 minutes of HCA at less than 20°C as herein used is known to be less tolerated by pigs than by humans, but these conditions provided a valuable and extremely severe test for any drug or method that is claimed to be potentially effective in this setting. In this regard, mortality represents still the major end point in the laboratory and in the clinical setting and, in turn, ischemic brain injury appears to be a major determinant of postoperative survival. All the animals included in this series survived after extubation, and the autopsy findings excluded any technical errors or other primary cause of death other than brain injury (eg, pulmonary or cardiac complications). Electroencephalogram burst recovery rates, histopathologic findings, and behavioral scores provided further evidence of impaired outcome in animals with major brain injury. However, even if the brain is the most vulnerable organ, other organs may significantly suffer during a 75-minute period of HCA leading to impairment of their function, which can affect postoperative outcome. The observation of increased survival among animals undergoing leukocyte filtration, therefore, confirms the importance of a global protection under such extreme conditions, and, in turn, the validity of such a severe experimental test.
In conclusion, this study provided further evidence of the beneficial effects of the Na+-channel blocker lamotrigine in mitigating brain injury after HCA. The combined use of lamotrigine and leukocyte filtration may further improve the survival outcome after HCA.
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Acknowledgments |
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We thank Janne Heikkinen, MS, Timo Kaakinen, MS, Erkka Rönkä, MS, Seija Seljänperä, RN, Veikko Lähteenmäki and Kauko Korpi, RN, for technical assistance.
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