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Departments of *Anesthesiology,
Surgery, and
Neurobiology, Duke University Medical Center; and
Duke University School of Medicine, Durham, North Carolina
Address correspondence and reprint requests to David S. Warner, MD, Box 3094, Duke University Medical Center, Durham, NC 27710. Address e-mail to warne002{at}mc.duke.edu
| Abstract |
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IMPLICATIONS: Rats were subjected to temporary focal cerebral ischemia and allowed to recover for 3 days or 2 wk. There was no effect of intraischemic N2O on histologic or behavioral outcome at either recovery interval, whereas the N-methyl-D-aspartate antagonist dizocilpine caused persistent improvement in both outcome measures.
| Introduction |
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The interaction between N2O and ischemic/hypoxic brain injury has been the focus of several studies (813). Conclusions have varied with respect to the effect of N2O on ischemic outcome. N2O has properties that might cause either an increase or decrease in ischemic brain injury. N2O increases the cerebral metabolic rate (14,15), and this might unfavorably balance the intraischemic metabolic substrate supply and demand. Hyperbaric N2O causes cytosolic vacuolizations of the cingulate gyrus (2). The addition of normobaric N2O to isoflurane and midazolam augments perinatal neuronal apoptosis in the rat, resulting in delayed behavioral abnormalities (16). At the same time, ischemia-induced increases in extracellular glutamate are widely believed to contribute to excitotoxic brain damage (17). Antagonism of the glutamatergic NMDA receptor with a variety of pharmacologic compounds provides at least a transient reduction in cerebral necrosis after an ischemic insult (1820). Thus, the NMDA antagonism properties of N2O may serve to improve ischemic outcome. These findings have caused renewed interest in examining the neuroprotective properties of N2O (13).
One dilemma in the study of N2O is its relatively low potency, such that when used alone, N2O does not offer sufficient anesthesia to be the sole anesthetic in most laboratory models of ischemic brain injury. As a result, the effects of N2O are typically assessed when N2O is added to a different anesthetic (9,10,12). This prevents direct and specific assessment of the effect of N2O on ischemic brain. Advances in animal modeling now allow the study of N2O alone under conditions of a focal cerebral ischemic insult. We hypothesized that N2O would have no effect on ischemic brain damage in rats subjected to transient middle cerebral artery occlusion (MCAO). To ensure the sensitivity of our model, we simultaneously examined the effects of dizocilpine, a selective NMDA receptor antagonist (21), by using a dose previously demonstrated to reduce ischemic brain injury when outcome was assessed at 1 wk postischemia (22). Given recent evidence that anesthetic protection against focal ischemic brain injury may be transient (23), outcome was observed at both 3 and 14 days postischemia. Finally, we explored elements of a novel neurologic scoring system designed to evaluate the recovery of rats from MCAO.
| Methods |
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Rats were anesthetized in a chamber with 5% isoflurane in 100% oxygen. The trachea was intubated, and the lungs were mechanically ventilated (30% oxygen/balance nitrogen). The inspired isoflurane concentration was reduced to 1%2%, and animals were prepared for MCAO by using modifications of the techniques described by Memezawa et al. (24) and Zea Longa et al. (25). Surgery was performed with aseptic technique, and all surgical fields were infiltrated with 1% lidocaine. The tail artery was cannulated and used to monitor arterial blood pressure and sample blood. The tail vein was cannulated for drug infusion.
A flexible thermistor (Mon-a-therm® Skin; Mallinckrodt, St. Louis, MO) was calibrated against a 37.5°C water bath. The probe was then percutaneously placed beneath the right temporal muscle adjacent to the skull and sutured to the skin. A silicon harness (Covance Infusion Harness; CIH95; Instech Solomon, Plymouth Meeting, PA) was placed on the rat. The thermistor wire was connected to the harness, which was then passed through a flexible coil attached to both the harness and a swivel commutator (Model SL2C 2 channel; Plastic Ones, Roanoke, VA). Pericranial temperature was servocontrolled (YSI Model 73ATA; YSI Inc., Yellow Springs, OH) at 37.5°C ± 0.2°C by surface heating and cooling (heating lamp or chilled fresh gas flow/ice) from surgery onset until 20 h postischemia. A strip chart record of pericranial temperature was made during this time.
A midline ventral cervical skin incision was made, and the right common carotid artery was identified. The external carotid artery was isolated, and the occipital, superior thyroid, and external maxillary arteries were ligated and divided. The internal carotid artery was dissected distally until the origin of the pterygopalatine artery was visualized. After surgical preparation, a 30-min interval was allowed for physiological stabilization. Heparin (50 IU) was given IV.
Physiologic values, including arterial carbon dioxide and oxygen partial pressures and arterial pH, were measured 10 min before MCAO onset, 45 min after MCAO onset, and 15 min after the onset of reperfusion. Hematocrit was determined 10 min before MCAO onset and 15 min after the onset of reperfusion. The blood glucose concentration was determined 10 min before MCAO onset. Mean arterial blood pressure (MAP) was continuously monitored until 15 min after reperfusion.
To achieve MCAO, a 0.25-mm-diameter nylon filament coated with silicon (0.38 mm diameter) was inserted into the external carotid artery stump and advanced 1819 mm from the carotid artery bifurcation into the internal carotid artery. The filament was secured, the wound was closed, and isoflurane (approximately 1.2%1.3% end-tidal concentration) was abruptly discontinued. A timer was started at MCAO onset. The animals were randomly separated into the following 3 conditions: 1) nitrogen group, 70% nitrogen/30% oxygen plus vehicle (0.6 mL of 0.9% NaCl); 2) N2O group, 70% N2O/30% oxygen plus vehicle; or 3) dizocilpine group, 70% nitrogen/30% oxygen plus dizocilpine ((±)MK-801; Sigma Chemical Co., St. Louis, MO; 0.25 mg/kg in 0.6 mL of 0.9% NaCl). Dizocilpine or vehicle was given IV over 2 min, and the tail vein catheter was removed.
The trachea was extubated after recovery of spontaneous ventilation and the righting reflex. Time to extubation was recorded. The animals were immediately placed in a clear acrylic box, and the assigned respiratory gas mixture was continued. During this interval, the concentrations of N2O and oxygen within the acrylic box were continuously monitored with a medical gas analyzer. A port in the box allowed the swivel system to be exteriorized to the commutator, allowing free movement of the animal within the box. All rats underwent 75 min of MCAO.
To terminate MCAO, 5% isoflurane was rapidly introduced into the respiratory gas mixture. N2O or nitrogen was discontinued and replaced with 100% oxygen. The rats were removed from the box, and a snout cone was placed. Inspired isoflurane was reduced to 1.5%. The neck wound was opened and the occlusive filament removed. The tail artery catheter was removed 15 min after reperfusion. All wounds were infiltrated with 1% lidocaine and closed with sutures. Isoflurane was discontinued, and the animals were allowed to awaken. Animals were placed in an acrylic animal enclosure (MTANK/W; Instech Solomon) and breathed 40% oxygen in room air for the first 3 h after reperfusion and then room air for 17 h. Free access to water and food was allowed. After 20 h, the animals were removed from the enclosure and briefly anesthetized with isoflurane to remove the harness and thermistor. The animals were returned to their cages for further recovery.
Two experiments were performed contemporaneously. These experiments were identical in all aspects, with the following exceptions: in Experiment 1 (n = 16 per group), rats underwent neurologic evaluation at 3 days after MCAO, and cerebral infarct size was measured. In Experiment 2 (n = 16 per group), rats underwent neurologic evaluation at both 3 and 14 days after MCAO. After the 14-day neurologic evaluation, infarct size was measured.
The neurological scoring system was derived by evaluating four different functions (general status, simple motor deficit, complex motor deficit, and sensory deficit) by combining elements from several previously described scoring systems (Table 1) (2629). The score given to each animal at the completion of the testing (by an observer blinded to group assignment) was the sum of all 4 individual scores: 0 was the minimum (best) score, and 48 was the maximum (worst) score.
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Infarct volume was measured by using the method of Swanson et al. (30). Serial quadruplicate 20-µm-thick coronal sections were taken by using a cryotome at 660-µm intervals over the rostral-caudal extent of the infarct. The sections were dried and stained with hematoxylin and eosin. A representative section from each 660-µm interval was digitized with a video camera controlled by an image analyzer (M2 Turnkey System; Imaging Research, St. Catharines, Ontario, Canada). The image of each section was stored as a 1280 x 960-pixel matrix and displayed on a video monitor. With the observer blinded to experimental conditions, the following regions of interest (ROI) were cursor-outlined: noninfarcted ipsilateral cerebral cortex, noninfarcted ipsilateral subcortex, contralateral cerebral cortex, and contralateral subcortex. The area within each ROI (square millimeters) was determined by automated counting of the calibrated pixels contained within the ROI. Ipsilateral noninfarcted cortex and subcortex areas were subtracted from the corresponding contralateral ROI values. Infarct volumes (cubic millimeters) were computed as running sums of subtracted infarct area multiplied by the known interval (e.g., 660 µm) between sections over the rostral-caudal extent of the infarct calculated as an orthogonal projection.
Experiments 1 and 2 were analyzed as independent events. One-way analysis of variance was used to compare physiologic values, total neurologic scores, and cortical, subcortical, and total infarct volumes among groups. When indicated by a significant main effect, post hoc testing was performed with the Scheffé test. A priori, P < 0.05 was considered significant. Because our primary dependent variables were limited to total neurologic score and total infarct volume for each experiment, a correction for multiple comparisons was not made. Linear regression (simple and multiple) was used to evaluate the relationship between total infarct volume and general, motor, and sensory evaluations and the combined effects of the different categories (collapsed across treatment groups and experiments). Values are mean ± SD.
| Results |
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The time to tracheal extubation after MCAO onset was different among groups in both experiments (Experiment 1: nitrogen, 10 ± 3 min; N2O, 19 ± 6 min; dizocilpine, 12 ± 4 min; P < 0.001). Values for Experiment 2 were similar. After extubation, animals in all groups were active, demonstrating rearing and circling behavior throughout MCAO, although both the N2O and dizocilpine groups appeared subdued.
Physiologic values are presented in Table 2. A main effect for group was present for body weight at both 3 days (P = 0.03) and 14 days (P = 0.006). At 14 days, post hoc testing revealed heavier body weights in the dizocilpine group versus the N2O group (P = 0.006). There also was a main effect for MAP at 45 min after the onset of MCAO in Experiments 1 (P = 0.01) and 2 (P = 0.001). In Experiment 1, intraischemic MAP was greater in the dizocilpine versus the nitrogen group (P = 0.014). In Experiment 2, intraischemic MAP was greater in the dizocilpine group versus both the nitrogen (P = 0.002) and N2O (P = 0.03) groups. There were no differences among groups for the remaining physiologic values.
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| Discussion |
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The goal of this study was not specifically to compare the effects of N2O and dizocilpine. Instead, dizocilpine was used as a positive control in the case that no effect of N2O was observed. This ensured that our model would be sufficiently sensitive in this iteration to detect an effect on outcome should it be present. However, because of information that N2O provides biologically relevant antagonism of the NMDA receptor (2,3), comparison with a selective NMDA receptor antagonist, such as dizocilpine (21), seemed appropriate. Dizocilpine produced a major and sustained improvement in both neurologic function and infarct size relative to both the nitrogen and N2O groups. This indicates that had an N2O effect been present, it would have been detected. The effect of dizocilpine was confounded by higher intraischemic MAP values compared with the nitrogen group in Experiments 1 and 2. We did not attempt to manipulate MAP values during this experiment. The extent to which this difference (approximately 1520 mm Hg) contributed to the protective effect of dizocilpine is not known, although previous work in the rat has shown that intraischemic MAP can modulate the volume of tissue at risk for infarction (31).
We do not know the relative magnitude of NMDA receptor antagonism induced by N2O and dizocilpine given the doses used in this experiment. The dizocilpine dose of 0.25 mg/kg IV was selected to ensure a positive control on the basis of prior research (22). To some extent, the anesthetic potency of the two drugs can be derived by considering the effects on minimum alveolar anesthetic concentration (MAC). The MAC for N2O has been reported to range from 1.5 to 2.2 atm (32,33). Thus, 70% N2O represented approximately 0.3 MAC. The effect of dizocilpine on MAC in the rat is complex with respect to spinal versus central sites of action (34). This limits our ability to define equipotency of the doses of N2O and dizocilpine used in this experiment. Nevertheless, MAC-reduction studies with dizocilpine indicate that doses in the range used in our study (i.e., 0.25 mg/kg) result in a 30%50% reduction in MAC (47). This suggests at least gross anesthetic equipotency for the doses of N2O and dizocilpine used in this experiment. However, the anesthetic/analgesic effect of N2O most likely involves mechanisms in addition to NMDA receptor antagonism (1,3537), making it likely that the NMDA antagonism induced in our experiment by N2O was substantially less than that caused by dizocilpine 0.25 mg/kg.
The results of this experiment are consistent with a previous report from our laboratory that found no effect of N2O on outcome from MCAO in the rat (10). In that study, rats were subjected to MCAO while anesthetized with a pentobarbital dose sufficient to cause sustained electroencephalographic burst suppression. The addition of 70% N2O to pentobarbital had no effect on short-term ischemic outcome. In contrast, Hoffman et al. (11) observed that the addition of N2O to fentanyl in rats subjected to hemispheric incomplete global ischemia caused worsened neurologic function at three days postischemia (histologic injury was not assessed). Because pericranial temperature and MAP were controlled during ischemia, those results must be considered as valid. Perhaps the difference in results between the study of Hoffman et al. (11) and our study can be attributed to differences in models (e.g., focal versus global ischemia). Alternatively, in the study by Hoffman et al. (11), rats were severely hyperglycemic during ischemia, and the extent to which this known determinant of ischemic outcome interacted with the addition of N2O is unknown.
Recently, David et al. (13) examined effects of N2O in a rat model of transient MCAO. In that study, ischemia occurred during halothane anesthesia. After ischemia, the animals were awakened and randomized to breathe room air, 75% N2O, or 50% or 75% xenon for the first three hours after reperfusion. Both N2O and xenon reduced cortical infarction by approximately 70% compared with room air. These startling results must be considered with skepticism in the context of the experimental protocol. There was no physiologic monitoring or control during N2O exposure, survival was limited to 24 hours, and no neurologic correlate was provided. Interventions in the postischemic phase with a purported major beneficial effect on ischemic outcome often fail to provide benefits when either temperature control or long-term outcome analysis is used (3840).
We continue to try to improve the neurologic evaluation of MCAO outcome because neurologic outcome is the primary dependent variable in clinical stroke trials. Improved assessment of therapeutic effects on neurologic outcome might lead to improved ability of preclinical studies to predict clinical efficacy. The MCA territory includes both the motor and sensory cortex. This suggests that inclusion of a sensory examination might improve the association between neurologic evaluation and cerebral infarct size. Further, we have performed numerous studies with simple motor scoring systems that provide categorical data, usually on a scale of 03. Although this crude assessment has provided statistically significant correlations with infarct size, we postulated that simultaneous assessment of more complex motor tasks would improve the validity of the neurologic assessment. In this experiment, we found that observation of the general status of the rat (e.g., activity) was a poor predictor of infarct size. Both simple and complex motor assessments provided statistically significant associations with infarct size, but the R2 value was substantially improved when the assessments were combined. Addition of the sensory element did little to further improve this association, perhaps because of the close proximity of the sensory and motor cortices within the MCA flow distribution. The data from this experiment, therefore, indicate that a comprehensive motor examination provides an improved association with lesion size compared with simple categorical assessment. Further improvements might include the assessment of hippocampal function, which is also injured with MCAO (41).
In conclusion, we compared the outcome from transient focal cerebral ischemia in rats breathing 70% N2O or 70% nitrogen during the ischemic insult. N2O altered neither neurologic function nor cerebral infarct size when assessed at either 3 or 14 days postischemia. In contrast, treatment with the NMDA receptor antagonist dizocilpine markedly improved outcome at both times. Although the effect of dizocilpine was temperature independent, we cannot exclude the possibility that the greater perfusion pressure associated with dizocilpine treatment contributed to the improved ischemic outcome. These data indicate that the NMDA receptor antagonism effects of N2O are insufficient to alter focal ischemic outcome.
| Acknowledgments |
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The authors are grateful for the expert technical assistance provided by Ann D. Brinkhous, Carla Calvi, MS, and Gary W. Massey, BS, MBA, Department of Anesthesiology, Duke University Medical Center.
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