Propofol Anesthesia Increases Dopamine and Serotonin Activities at the Somatosensory Cortex in Rats: A Microdialysis Study

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1 GENERAL ARTICLES Propofol Anesthesia Increases Dopamine and Serotonin Activities at the Somatosensory Cortex in Rats: A Microdialysis Study Ming-Hwang Shyr, MD, PhD*, T. H. Tsai, PhDS, Chen-Hsien Yang, MD*, Han-Ming Chen, Mm, Ho-Fu Ng, MD*, and Peter I?. C. Tan, MD* Departments of *Anaesthesia and tsurgery, Chang Gung Memorial Hospital and Chang Gung College of Medicine and Technology, Taoyuan, Taiwan, Republic of China; and SNational Research Institute of Chinese Medicine, Taipei, Taiwan, Republic of China We sought to estimate the activities of dopamine and serotonin in animals receiving propofol anesthesia. The in uiuo microdialysis technique was used in Sprague-Dawley rats (n = 6) to measure the major metabolites of dopamine and serotonin, i.e. 3,4- dihydroxyphenylacetic acid (DOPAC), homovanillic acid (4-hydroxy-3-methyphenylacetic acid; HVA) and 5-hydroxy indole acetic acid (5-HIAA) in the somatosensory cortex. We also measured the levels of propofol in the brain and blood by microdialysis sampling in another group of rats (n = 6). During the experiment, the rat was infused intravenously (IV) with propofol at a rate of 10 mg * kg-. h-i for 60 min and 60 mg. kg- - h-i for 40 min. We found that IV infusion of propofol at a rate of 60 mg. kg- * h- significantly increased DOPAC, HVA, and 5-HIAA. We also determined that these changes correlated well with propofol levels in the brain and blood. We concluded that anesthetic doses of propofol increased the functional activities of dopamine and serotonin in the cortex. These increases correlate well with propofol levels in the cortex and blood. (Anesth Analg ) P ropofol has been extensively used clinically as a hypnotic and nonhypnotic drug. Although the therapeutic applications of propofol are diverse, its mechanisms of action are largely unknown. Many studies have implied that propofol may interact with dopamine receptors (l-5) and, perhaps, serotonin receptors (1,6,7). However, there is no direct evidence that propofol alters the release of these two neurotransmitters. The aim of this study was to apply the in viva microdialysis technique to measure the major metabolites of dopamine and serotonin, i.e. 3,4- dihydroxyphenylacetic acid (DOPAC), homovanillic acid (4-hydroxy-3-methyphenylacetic acid; HVA), and 5-hydroxy indole acetic acid (5-HIAA), in the brain to This work was supported in part by research grants NSC B from the National Science Council, Republic of China; NMRP561 from the Chang Gung Memorial Hospital to PPCT and MHS; and CMRP613 from the Chang Gung Memorial Hospital to MHS. Accepted for publication February 12, Address correspondence and reprint requests to P. P. C. Tan, MD, Department of Anaesthesia, Chang Gung Memorial Hospital, 5 Fu-Shin St., Kwei-Shan, Taoyuan 33333, Taiwan, Republic of China. Address to anool@adm.cgmh.com.tw. estimate the activities of dopamine and serotonin in animals receiving propofol anesthesia. Using a microdialysis sampling technique, we also measured the propofol levels in the brain and blood dialysates and correlated them with changes in these biogenic amines. Methods The procedures used in this study were approved by our institutional experimental animal ethics committee. Experiments were performed in adult, male Sprague-Dawley rats ( g). The animals were housed in an animal room with a 12:12-h light-dark cycle (lights on 8:OO AM-S PM) and an ambient temperature of 22 2 l C, with food and water available ad libitum. Animals (n = 6) were initially anesthetized with pentobarbital (40 mg/ kg intraperitoneally). The ensuing narcosis, which lasted min (8), allowed us sufficient time (no more than 30 min) to complete the routine surgical and experimental preparations without the need for supplemental administration of pentobarbital. The right femoral vein was cannulated for 1344 Anesth Analg by the International Anesthesia Research Society /97/$5.00

2 ANESTH ANALG SHYR ET AL drug administration. The head of the animal was fixed in a stereotaxic apparatus (Kopf 900; David Kopf Instruments, Tujunga, CA), and a microdialysis probe (active length 2 mm, diameter 0.5 mm; CMA/ Microdialysis AB, Stockholm, Sweden) was implanted in the somatosensory cortex (4 mm lateral and 1 mm posterior to the bregma) (9). The animals were allowed to spontaneously breathe room air, and their rectal temperature was maintained at 37 C with a heating pad. To provide analgesia during the experiment, all incision sites and the bilateral auditory canals were infiltrated with 0.25% bupivacaine. Two hours after the initial injection of pentobarbital, the animals were infused with intravenous (IV) propofol at a rate of 10 mg * kg-l. h- for 1 h, followed by 60 mg * kg- * h-l for 40 min. Administration of propofol was then terminated, and the animals were observed for another 60 min. During this period, the cortex was perfused with lactated Ringer s solution (147 mm Na+, 4.0 mm K+, 2.2 mm Ca +) at a rate of 1.2 pl/ min. Twenty microliters of dialysate was assayed with on-line high-performance liquid chromatography coupled with an electrochemical detector (10) to determine the concentrations of DOPAC, HVA, and 5-HIAA simultaneously. Before propofol was initiated, the dialysis probe was perfused with lactated Ringer s solution for at least 60 min to establish equilibrium. In another experiment, we repeated the above procedure to determine the concentrations of propofol in the somatosensory cortex and blood and correlated those with alterations in biogenic amines. Since there was no commercially available probe, we constructed our own dialysis probe and implanted it into the right jugular vein to the level of the right atrium to measure the blood propofol level. The exchange component of the probe was a regenerated cellulose dialysis membrane (Spectra/ Por RC; Spectrum Medical Industries, Inc., Houston, TX; 200 pm inner diameter, 220 pm outer diameter, molecular weight cutoff 6,000 D). The active length was 10 mm. The rat was injected IV with heparin 1 U/g of body weight to prevent blood clotting on the dialysis membrane of the IV probe. During IV infusion of propofol (10 or 60 mg * kg- * h-l), the microdialysis probes in th.e brain and blood stream were perfused with lactated Ringer s solution at a rate of 1.2 pl/min. Twenty microliters of dialysate was directly assayed offline with high-performance liquid chromatography coupled with a fluorescence detector as modified from the method of Plummer (11) to determine the level of propofol. The probe was calibrated by inserting it into a tube containing 100 nm biogenic amines and 50 nm propofol, respectively. The perfusion media and pumping flow were the same as in the above experiments. The Table 1. Measured and Corrected Basal Values of Cortical DOPAC, HVA, and 5-HIAA DOPAC HVA 5-HIAA WI (W wf) Measured values Mean probe recovery Corrected values 72.8 t t 25.8 Data are mean? SEM. DOPAC = 3,4-dihydroxyphenylacetic acid; HVA = 4.hydroxy-3. methyphenylacetic acid; 5-HIAA = 5-hydroxy indole acetic acid. Corrected value = measured value/mean probe recovery. probe recovery was calculated by dividing the concentrations in the dialysate by the concentration in the tube. All data are mean + SEM. Repeated-measurement analysis of variance was used to compare the means within the group. Post hoc multiple comparisons were performed using the Dunnett s test. A P value less than 0.05 was considered statistically significant. The half-life of propofol was determined by first-order linear regression. We calculated the time required to decrease the peak level of propofol to its half-value. Results The measured and correlated basal values of DOPAC, HVA, and 5-HIAA are given in Table 1. The same probe was of high consistency in vitro for at least 4 h. However, the recovery of each probe for measuring the same biogenic amine varied from 32% to 47%. The corrected values were calculated from measured values divided by mean probe recovery. DOPAC and HVA remained unchanged after the initiation of IV propofol at a rate of 10 mg * kg-l * h- (Fig. 1A). At a more rapid rate (60 mg * kg- * h-l), DOPAC and HVA were significantly increased approximately twofold. Termination of propofol administration rapidly restored DOPAC and HVA to their basal values. Conversely, 5-HIAA increased to approximately 50% above the basal value at the end of propofol infusion at 10 mg. kg- *h-l and remained largely unchanged, even when the infusion rate increased to 60 mg * kg- * h-l. Like DOPAC and HVA, 5-HIAA rapidly returned to its basal value when propofol administration was stopped. After the initiation of propofol at the slower infusion rate, the propofol level increased rapidly and remained relatively constant for 60 min. At the more rapid rate (60 mg * kg- * h-l), the propofol levels in brain and blood increased rapidly and reached their peaks at the end of administration (Fig. 1B). The measured and corrected propofol levels 40 min after propofol administration are given in Table 2. These alterations paralleled the changes in DOPAC, HVA, and

3 1346 SHYR ET AL. ANESTH ANALG Table 2. Measured and Corrected Propofol Levels 40 Minutes After Propofol Infusion at 60 mg * kg- * h- h F e Co iii g 3 IOO OV B 0 DOPAC 0 HVA v 5HIAA TIME (min) o Blood I,,,,,,,,, TIME (min) Figure 1. A, Effect of propofol anesthesia on the temporal changes in 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (4- hydroxy-3-methyphenylacetic acid; HVA) and 5-hydroxy indole acetic acid (5-HIAA) in dialysates. Propofol was infused at a rate of 10 mg * kg- * h-i at O-60 min and 60 mg/kg/h at min. Data are represented as percentages of the basal values. Values are mean t SEM, n = 6 animals per group. P < 0.05 compared with baseline for DOPAC, HVA, and 5-HIAA. B, Temporal changes of propofol concentrations in the brain and blood dialysates during propofol anesthesia. Values are mean 2 SEM, n = 6 animals per group. 5-HIAA (Fig. 1A). The half-life of propofol in the brain and blood was 29.8 and 31.4 min, respectively. Discussion Propofol has been extensively used clinically as a hypnotic and nonhypnotic drug. It acts largely by unknown mechanisms. Like all other general anesthetics, Brain (nm) Blood (nm) Measure values Ifr 3.8 Mean probe recovery (%) Corrected value t k 8.8 Corrected value = measured value/mean probe recovery. no specific site of action has yet been identified for propofol anesthesia. Propofol may interact directly with dopamine (l-5) and serotonin receptors (1,6,7) to produce emesis. However, whether propofol alters the release of dopamine and serotonin in the brain is unclear. We believe our technique resolves this issue. Measurements by in vim microdialysis of the major metabolites of dopamine and serotonin, i.e., DOPAC, HVA, and 5-HIAA, in the somatosensory cortex have been used as indices of functional activities of dopamine and serotonin (12). We found that anesthetic doses of propofol significantly increased dopamine and serotonin activities in the cortex. These increases correlate well with propofol levels in the cortex and blood. Propofol may have bidirectional actions on dopamine system. Appadu et al. (1) and DiFlorio (2) suggested that propofol is an antagonist of the D,- dopamine receptor. Because of this property, general anesthesia with propofol is associated with a decreased incidence of early postoperative nausea and vomiting. Recently, propofol has been successfully used in the management of patients with Parkinson s disease, but it produces dyskinesia (4). In contrast, Hvarfner et al. (5) found that propofol has no effect on apomorphine-induced vomiting. Keita et al. (3) also suggested that propofol, in higher relevant clinical doses, might inhibit uptake of dopamine into striatal synaptosomes. Inhibition of dopamine uptake may cause accumulation of dopamine and its major metabolites, DOPAC and HVA, in the extracellular space. Our data indicate that propofol increases dopamine activity in the cortex, probably via increased release or decreased uptake of dopamine. Halothane increases dopamine release in the rat brain (13). Unlike halothane, which produces a high incidence of vomiting, propofol may produce antiemesis at subhypnotic doses, even when administration has been stopped for a long time. Our study indicates that DOPAC and HVA levels remained high for 40 minutes after the infusion was stopped. Brains of rats anesthetized with propofol have increased levels of metabolites of dopamine. Hence, the antiemetic effect of propofol may not be the result of propofol acting against dopamine receptors. Propofol may produce antiemesis by another mechanism.

4 ANESTH ANALG SHYR ET AL Clinically, propofol may also act directly as an antagonist on serotonin, especially 5-HT, receptors, or it may potentiate the action of serotonin antagonists to prevent emesis during chemotherapy (1,6). However, in vitro studies do not support this hypothesis. Appadu and Lambert (7) and Machu and Harris (14) found that in the clinical range, propofol did not bind to or alter the function of 5-HT, receptors. Our data indicate that propofol increases serotonin activity in the cortex. The change in 5-HIAA due to propofol administration was different from that of DOPAC and HVA. Propofol increased approximately 50% above its basal value at the end of the infusion at the concentration of 10 mg. kg-i. h-l. The increased amplitude was largely equal to that produced by propofol at 60 mg * kg- * hp. The effect lasted for only 20 minutes after the propofol infusion was terminated. Further study is required to differentiate the effects of propofol on 5-HIAA, DOPAC, and HVA metabolism. Our data and in vitro studies do not agree with the clinical implication that propofol antagonizes serotonin activity. It should be pointed out that there is no significant effect of pentobarbital on dopamine (15) and serotonin (16,17) activities in rats. Hence, changes in DOPAC, HVA, and 5-HIAA concentrations after propofol administration may not be due to the residual influence of pentobarbital. We previously used multiple physiologic variables to determine the effective anesthetic dose of propofol in rats (18-20). We suggested that propofol may produce blood pressure and electroencephalographic (EEG) suppression on the somatosensory cortex in rats by IV bolus administration (18) or IV infusion (19) in a dose-dependent manner. Only when a threshold concentration is reached does propofol produce effective antinociception and EEG synchronization or burst suppression (i.e., by definition, anesthesia)(20, 21). IV infusion of propofol at a rate of 60 mg * kg-* * h-i may establish effective anesthesia 30 minutes after the infusion (19-21). At the much lower rate of 10 mg * kg-r * h-l, propofol can only produce sedation, as indicated by EEG synchronization to the 6 and 0 bands (20). The blood level of propofol will never reach the threshold level of antinociception, at least in 90 minutes (20). In our study, we compared the levels of biological amines with propofol levels during propofol administration (Fig. 1A). We found that propofol significantly increased DOPAC, HVA, and 5-HIAA levels when the anesthetic dose was applied. Thus, our findings suggest that dopamine and serotonin are associated with propofol-elicited anesthesia. Like all other general anesthetics, no specific site of action has yet been identified for propofol anesthesia. Whether the changes of biogenic amines in the somatosensory cortex during propofol administration are generous phenomena for propofol anesthesia remains unclear and deserves further study. Because of its low molecular weight cutoff (~6,000 D) protein-bonded propofol cannot pass through the dialysis membrane. Only free propofol in the brain and blood entered across the membrane and was measured. In addition, because there was no commercially available probe long enough to be inserted into the level of the right atrium, which would represent the mixed venous concentrations of propofol, we measured the blood propofol concentration using selfconstructed probes. The discrepancy of recovery between the brain and blood probes (2.2% vs 43%)(Table 2) may be largely due to probe size and membrane character. By using brain tissue homogenization, we previously determined that propofol is evenly distributed in the brain and spinal cord (18). We also found that brain propofol concentrations are 7.8 times higher than plasma propofol concentration. By using a microdialysis technique, we found that brain and blood propofol levels correlate well during propofol administration, and the brain/blood ratio is roughly 10. Accordingly, the protein-binding fraction of propofol is similar in the brain and blood. One of the major benefits of propofol anesthesia is its good concentration-effect relationship. In targetcontrolled anesthesia, one can used blood propofol concentration as an index to predict the anesthetic depth of propofol (22). Whether free propofol concentrations in the brain correlates well with blood concentrations during propofol anesthesia is questionable. In this study, we found that propofol concentrations in the blood correlate well with those in the brain and that the half-lives of free propofol in the brain and blood were similar (30 minutes), which is close to the metabolic half-life (23) but not the clinical half-life of propofol. As in our previous studies (20,21), this suggests that a threshold level of propofol must be achieved to produce alterations of the biogenie amines and, thereafter, their biological effects. We conclude that propofol at an anesthetic dose significantly increases dopamine and serotonin activities in the cortex. These increases correlate well with propofol levels in the cortex and blood. References 1. Appadu BL, Strange PG, Lambert DG. Does propofol interact with D, dopaminereceptors? Anesth Analg 1994;~79: DiFlorio T. Is urouofol a dooamine I antagonist? lletterl. L 1 Anesth Analg 1993;77:2Ot?. 3. Keita H, Lecharny JB, Desmonts JM, et al. 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