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Departments of Anesthesiology,
*University of California, San Diego and Department of Veterans Affairs, Veterans Affairs Medical Center, San Diego; and
Loma Linda University, Loma Linda, California
Address correspondence and reprint requests to Piyush M. Patel, Department of Anesthesiology, Anesthesia Service 9125, VA Medical Center, 3350 La Jolla Village Dr., San Diego, CA 92161. Address e-mail to ppatel{at}ucsd.edu
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Abstract |
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Implications: Fentanyl is commonly used in surgical procedures in which there is a substantial risk of focal cerebral ischemia. Fentanyl did not affect cerebral injury produced by focal ischemia in the rat. The data suggest that, in doses that produce respiratory depression and muscle rigidity, fentanyl does not reduce the tolerance of the brain to a focal ischemic insult.
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Introduction |
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Accordingly, we conducted the present study to evaluate the effect of fentanyl administration on outcome after temporary focal cerebral ischemia in rats. The effect of fentanyl was compared with that of isoflurane. Animals that were allowed to awaken during the ischemic interval served as controls.
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Methods |
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Wistar-Kyoto rats (Simonson Laboratories, San Diego, CA) weighing 275–325 g were fasted overnight. Access to water was provided. The rats were anesthetized with an inspired concentration of 5% isoflurane (Ohmeda, Liberty Corner, NJ). After induction of anesthesia, the animals' tracheas were intubated, and their lungs were mechanically ventilated with a gas mixture of 30% oxygen/70% nitrogen. The concentration of isoflurane was reduced to 2.5% end-tidal (measured by advancing a 22-gauge needle through the endotracheal tube to the carina). A 5-mm dorsal transverse incision was made 1 cm caudal to the ears. A pericranial temperature probe (Mona-a-Therm; Mallinckrodt, St. Louis, MO) was tunneled via the neck incision, and it was inserted between the left temporalis muscle and the skull. The temperature probe was then sutured in place. Thereafter, pericranial temperature was servocontrolled to 37.0 ± 0.2°C (Model 73A; Yellow Springs Instruments, Yellow Springs, OH) with the combination of a water-heated blanket and an overhead heat lamp. The tail artery was cannulated with PE-50 tubing. The mean arterial pressure (MAP) was monitored continuously. The right external jugular vein was exposed via a midline pretracheal incision and was cannulated with PE-50 tubing. Platinum needle electrodes were inserted in a biparietal configuration, and the electroencephalogram (EEG) (Grass Instruments, Quincy, MA) was monitored continuously.
The animals were prepared surgically for the occlusion of the middle cerebral artery according to the technique of Zea-Longa et al. (2). The right common carotid artery was exposed via a midline pretracheal incision. The vagus and sympathetic nerves were carefully separated from the artery. The external carotid artery was ligated 2 mm distal to the bifurcation of the common carotid artery. The pterygopalatine artery was then ligated approximately 1 mm distal to its takeoff. The common carotid artery was then permanently ligated. Via a small arteriotomy, a 0.25-mm diameter nylon monofilament previously coated with silicone was inserted into the proximal common carotid artery.
The animals were then allocated randomly to one of three experimental groups. In the fentanyl group, 50 µg/kg undiluted fentanyl (Elkins-Sinn Incorporated, Cherry Hill, NJ) was administered IV over a period of 10 min. Thereafter, a continuous infusion of fentanyl was initiated at a rate of 50 µg · kg-1 · h-1. Simultaneously, the concentration of isoflurane was reduced to 1.1% end-tidal. In the awake and isoflurane groups, the concentration of isoflurane was reduced to 1.1% end-tidal (approximately 1 minimum alveolar anesthetic concentration [MAC]) (3). These animals received an IV infusion of isotonic sodium chloride solution at a rate of 4 mL · kg-1 · h-1. The animals were left undisturbed for 30 min. During this time, arterial blood gas tensions were measured. Ventilation variables were adjusted to maintain PaCO2 in the range of 35–40 mm Hg. Serum glucose and hematocrit were measured and recorded.
After the 30-min equilibration period, focal cerebral ischemia for 90 min was induced by advancing the monofilament into the anterior cerebral artery via the common carotid artery to a distance of 18–20 mm from the bifurcation of the common carotid artery. Advancement of the filament was halted when mild resistance was encountered. The monofilament was then secured. The pretracheal wound was closed with a suture. All wounds were subsequently infiltrated with 0.25% bupivacaine (Abbott Laboratories, Chicago, IL; total dose 0.5 mg).
In the awake group, isoflurane administration was discontinued. On resumption of spontaneous ventilation, mechanical ventilation was discontinued, and the endotracheal tube was removed. The EEG electrodes were also removed. Thereafter, the animals were transferred to a heated and humidified incubator through which oxygen was continuously flushed. The animals were anesthetized briefly with isoflurane at the end of the ischemic period. The pretracheal incision was reopened, and the monofilament was removed from the common carotid artery. The wound was resutured and the animals were allowed to awaken.
In the isoflurane group, the concentration of isoflurane was maintained at 1.1% end-tidal throughout the ischemic interval. The EEG was monitored continuously. The monofilament was removed from the common carotid artery at the end of the 90-min ischemic interval. The animals were then allowed to awaken.
In the fentanyl group, isoflurane administration was discontinued after the initial suture closure of the pretracheal incision. The lungs were mechanically ventilated with a gas mixture of 30% oxygen/70% nitrogen. In 15 animals, spontaneous ventilation resumed. In these animals, mechanical lung ventilation was temporarily stopped. After 5 min, arterial blood gas tensions were measured. All animals had a PaCO2 within a range of 38–45 mm Hg. The tracheas of these animals were then extubated, and the EEG electrodes were removed. Arterial gas tensions were measured again approximately 30 min later to determine whether the animals' ability to breathe was adequate. In the remaining 11 animals, spontaneous respiratory efforts were not observed. In these animals, mechanical ventilation of the lungs was maintained, and the EEG was monitored continuously throughout the ischemic interval. At the end of the ischemic period, the infusion of fentanyl was discontinued. The animals were reanesthetized briefly with isoflurane. The monofilament was removed from the common carotid artery. The pretracheal wound was closed with suture. The EEG electrodes were removed, and the animals were then allowed to awaken. In the animals that required mechanical ventilation of the lungs, the endotracheal tubes were removed within 45 min after the onset of reperfusion.
Pericranial temperature was servocontrolled at 37.0 ± 0.2°C throughout the ischemic interval. During the recovery period, the pericranial temperature was recorded at 1-h intervals for 4 h. Thereafter, the temperature probe was removed. In addition, the morning rectal temperature was measured daily for 4 days after ischemia.
The animals were killed 7 days after ischemia. The rats were anesthetized with isoflurane for transcardiac perfusion with 200 mL of heparinized saline, followed by 200 mL of buffered 4% paraformaldehyde. The animals were decapitated, and the brains were left in situ at a temperature of approximately 4°C for 24 h. Thereafter, the brains were carefully removed, immersed in fixative, and refrigerated at a temperature of approximately 4°C for another 24 h. The brains were then prepared for histologic analysis. After dehydration in graded concentrations of ethanol and butanol, the brains were embedded in paraffin. Coronal sections (8 µ) at intervals of 0.75 mm were prepared and stained with hematoxylin and eosin.
Injury to the brain was evaluated by image analysis using National Institutes of Health Image 1.60 software and a computer. The analysis was performed by two observers who had no prior knowledge of the experimental groups. The area of injury to the subcortex and to the cortex was determined. The total volume of injury was determined by integration of the area of injury in each section according to the technique of Swanson et al. (3).
The physiologic values were evaluated by using a repeated-measures analysis of variance (ANOVA) (Statview 4.0; Abacus Concepts, Berkeley, CA). The volume of tissue injury was analyzed by using a single-factor ANOVA. When the ANOVA identified significant differences, post hoc Scheffé's tests were used for intergroup comparisons. A P value <0.05 was considered to be statistically significant. All data are presented as mean ± SD.
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Results |
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The physiologic variables are presented in Table 1. Baseline MAP and heart rate (HR) were similar in the three groups. During ischemia, the MAP and HR were significantly greater in the awake and fentanyl groups than in the isoflurane group. At the end of the 90-min ischemic period, there were no differences in MAP and HR among the groups.
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The temperatures (pericranial temperature before and during ischemia and for 4 h after ischemia and rectal temperature 1, 2, 3, and 4 days after ischemia) of the animals in the groups are presented in Figure 1. Temperature differences among the groups were not observed at any time. Hyperthermia after ischemia was not evident in any group.
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The total infarct volumes in the three groups are presented in Figure 2. In the subcortex, the infarct volumes were similar among groups (P = 0.25). The cortical infarct volume in the isoflurane group was less than that in the fentanyl and awake groups (P = 0.01). There was no difference in infarct volume between the awake and fentanyl groups (P = 0.91).
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Discussion |
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The observation that fentanyl did not increase brain injury after cerebral ischemia seems to be inconsistent with the results reported by Kofke et al. (4). These investigators reported that fentanyl substantially increased neuronal injury (compared with a control state in which fentanyl had not been given) in animals subjected to severe forebrain ischemia. This was attributed in part to the increased brain metabolic rate produced by opioid-induced seizure activity.1Differences in the experimental approach and the model used can probably explain these conflicting results. In the study of Kofke et al., histologic injury was evaluated 18 h after ischemia. In the forebrain ischemia model, delayed neuronal death can occur for several days after ischemia (6). Therefore, the difference in injury between the fentanyl group and the control group may not have been apparent had the recovery period been extended from 4 to 7 days. In support of this argument, Morimoto et al. (1) have shown that, in a dose of 400 µg/kg (sufficient to cause seizure activity), fentanyl did not increase neuronal injury after forebrain ischemia. In that study, injury was evaluated 4 days after ischemia. The authors conclude that fentanyl did not produce any worsening of brain injury in the setting of global ischemia. In vitro data also indicate that fentanyl does not enhance ischemic neuronal injury. In hippocampal slices, fentanyl did not affect electrophysiologic recovery after anoxic injury, i.e., fentanyl was neither neurotoxic nor neuroprotective (7). The results of the present study are consistent with the findings of Morimoto et al. (1) and Charchaflieh et al. (7) in that they demonstrate an absence of a deleterious effect of fentanyl during focal ischemia.
The results of the present study must also be reconciled with the previous demonstrations of the beneficial effect of naloxone, a µ-receptor antagonist, in models of cerebral ischemia and injury. For example, naloxone improved neurologic recovery in a model of traumatic cervical (8,9) and thoracic spinal cord injury (10) and in a model of spinal cord ischemia (11). In addition, naloxone has also been shown to improve outcome in gerbil (12), dog (13), and cat (14) models of cerebral ischemia. Collectively, these studies suggest an important role for opiate receptors in the pathophysiology of ischemia. However, they do not conclusively prove the involvement of µ-receptors for two reasons. First, not all studies have demonstrated an improvement in neurologic outcome with naloxone administration. Naloxone has also failed to reduce ischemic neuronal injury in gerbils subjected to forebrain ischemia (15). Previous work from our group has also demonstrated that naloxone does not improve outcome in a rabbit spinal cord ischemia model (16). Second, the dose of naloxone required to produce a beneficial effect (>2 mg/kg) is far in excess of that required to antagonize µ-receptors (17). As a result, it has been suggested that the beneficial actions of naloxone are the result of its activity at opiate receptors other than µ-receptors.
Isoflurane anesthesia substantially reduced postischemic cerebral injury. The precise mechanism by which this reduction in injury was achieved is not clear. Uncontrolled release of glutamate during ischemia and the consequent excessive stimulation of postsynaptic glutamate receptors (excitotoxicity) plays a major role in the initiation of neuronal injury (18). Previous investigations have shown that isoflurane can inhibit the release of glutamate from anoxic brain slices (19) and from the cortex of rats subjected to incomplete forebrain ischemia (20). In addition, inhibition of postsynaptic glutamate receptors by isoflurane has also been demonstrated. In neocortical slices, isoflurane reduced neuronal depolarizing responses evoked by the application of glutamate and N-methyl-D-aspartate (NMDA) on dendrites (21). Isoflurane also reduced the frequency of NMDA receptor channel opening and the mean open time of the channel in response to stimulation by NMDA (22). The expected increase in intracellular calcium concentration in response to application of NMDA was reduced by isoflurane in cultured hippocampal cells (23) and in neocortical brain slices (19). Collectively, these data indicate that attenuation of excitotoxicity by isoflurane anesthesia may have contributed to the observed reduction in ischemic injury in the isoflurane group.
Another potential mechanism by which isoflurane may have reduced cerebral injury is by the reduction of the stress response during ischemia. There is a significant increase in the levels of circulating catecholamines (a marker of the stress response) during ischemia. Werner et al. (24) have demonstrated that suppression of ischemia-induced catecholamine release substantially reduces neuronal injury. Administration of exogenous catecholamines can, however, augment injury (24). It is probable that the stress response in the awake and fentanyl-sedated animals was greater than that in the isoflurane group. This may also have contributed to the smaller infarct volumes in the isoflurane group.
The observation that isoflurane reduced infarct volume after focal ischemia is at a variance with the results reported by Sarraf-Yazdi et al. (25). In a model of focal ischemia in the rat similar to that used in the present study, these investigators did not observe a statistically significant reduction in infarct volumes in animals anesthetized with 0.7% isoflurane compared with animals kept awake during ischemia. A trend toward smaller infarct volumes in the isoflurane-anesthetized animals, however, was apparent. A significant methodologic difference between the study of Sarraf-Yazdi et al. (25) and the present study is the dose of isoflurane that was administered to the animals during ischemia. In the former, a smaller dose of isoflurane (0.7%) was used than in the present study (1.1%, MAC). In doses of 1 MAC, isoflurane has been shown to result in NMDA receptor antagonism in vitro (see above). If NMDA antagonism is one of major mechanisms by which isoflurane reduces ischemic injury, the degree of NMDA receptor antagonism and, therefore, the degree of neuroprotection, would be expected to be greater in the present study. Our results are consistent with this argument. This argument also suggests that the ability of isoflurane to reduce focal cerebral injury may well be dose-related. This premise is worthy of further investigation.
The rationale for the amount of fentanyl given to the rats in this study needs clarification. In the study of Kofke et al. (26), fentanyl was found to produce neuronal injury (nonischemic brain) when given in doses >400 µg/kg. Based on these data, a dose of 400 µg/kg was chosen. However, pilot experiments revealed that, in a significant number of animals, muscular rigidity and seizure activity during ischemia resulted in acidemia (blood pH <7.30). The acidemia could have been reduced by the administration of muscle relaxants. However, in our opinion, this could be justified only if nitrous oxide had been administered in addition to the fentanyl. Because it was our intention to specifically evaluate the effect of fentanyl on postischemic neuronal outcome, we chose not to give nitrous oxide. As a pragmatic compromise, we reduced the total dose of fentanyl to 150 µg/kg. This dose produced clinical evidence of significant µ-receptor agonism (mild to moderate muscle rigidity, sedation, and hypoventilation) without evidence of seizure activity. Although mild acidemia did develop in the fentanyl group, the reduction in pH was relatively minor.
The results of this study are also of relevance to investigations of the pathophysiology of cerebral ischemia in general. Many investigative approaches require a control group that is anesthetized. Anesthetics that are most commonly used to anesthetize the "control" group are the volatile anesthetics (halothane in particular). There is a large body of evidence that indicates that volatile anesthetics reduce ischemic brain injury substantially (27–29). This makes the comparison of the effect of novel pharmacologic drugs on outcome after cerebral ischemia with an anesthetized control state difficult. A potential solution is the use of synthetic opioids such as fentanyl. Our results show that, in the dose administered in this study, fentanyl does not affect outcome after focal ischemia. Warner et al. (30) have also shown that nitrous oxide does not influence total infarct volume after focal ischemia. Therefore, a combination of fentanyl and nitrous oxide may provide an alternative anesthetic technique for studies in which an anesthetized control state is required.
In summary, isoflurane reduced ischemic brain injury after 90 min of temporary focal ischemia. This is consistent with previous reports in which the ability of volatile anesthetics to reduce ischemic brain injury was demonstrated. The infarct volume in the fentanyl-sedated animals was similar to that in the nonanesthetized awake group. These data indicate that, in a dose of 150 µg/kg, fentanyl does not materially affect brain injury after focal cerebral ischemia.
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Acknowledgments |
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Footnotes |
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