Electroencephalograph), and clinical Neurophysiology, 1990, 7 5 : 5 4 3 - 5 4 7
543
Elsevier Scientific Publishers Ireland, Ltd.
EEG03785
Acute effects of anticonvulsants on brain-stem auditory evoked potentials in rats G. Hirose, T. Chujo, S. Kataoka, J. Kawada and A. Yoshioka Department of Neurology, Kanazawa Medical University, Uchinada, Kahoku-gun (Japan) (Accepted for publication: 23 July 1989)
Summary Acute effects of various anticonvulsants on brain-stem auditory evoked potentials (BAEPs) in rats were tested. A high dose of phenytoin abolished all BAEP waves. Carbamazepine increased all 4 wave latencies. Phenobarbital and clonazepam increased the latencies of waves IV and IlL Therefore, if BAEPs are to be used as an ancillary test for brain death, the possibility of significant effects of high dosages of anticonvulsants on BAEPs must be considered..
Key words: Brain-stem auditory evoked potentials; Anticonvulsants; Phenytoin; Carbamazepine
Brain-stem auditory evoked potentials (BAEPs) are measures of electrical events generated in the auditory pathway of the brain-stem and are recorded from cephalic electrodes by the far-field averaging method. This technique has been used frequently to detect structural damage to the auditory pathways in the brain-stem caused by tumors, demyelination or ischemic diseases. In contrast, BAEPs have been reported to be normal in toxic and metabolic encephalopathies when the anatomical substrate of the brain-stem is not damaged (Starr and Achor 1975). This interpretation has been widely used in clinical neurology for diagnosing toxic and metabolic encephalopathies. We recently saw a patient with phenytoin (PHT) intoxication whose BAEP waves had almost disappeared (Hirose et al. 1986). This experience prompted us to study the acute effects of high doses of anticonvulsants on rat BAEPs to establish whether they affect BAEP waves.
Correspondence to: Genjiro Hirose, M.D., Ph.D., Department of Neurology, Kanazawa Medical University, Uchinada, Kahoku-gun, Ishikawa Prefecture 920-02 (Japan),
Methods Adult male Sprague-Dawley rats weighing 300350 g were anesthetized intraperitoneally with 40 m g / k g pentobarbital. The rats were ventilated mechanically with room air, if necessary. Rectal temperature was monitored with a thermistor and telethermometer and controlled at about 3 7 ° C with an electric heating pad. After anesthetization the baseline BAEPs of the controls were obtained. After trying several high dosages of the test anticonvulsants, the maximal non-lethal dose for each was established for our experimental conditions. Acutely intoxicated rats were produced by intravenous (i.v.) injections of P H T (dose: 80 m g / k g ) , ethosuximide (ESM, dose: 250 m g / k g ) , sodium valproate (VPA, dose: 250 m g / k g ) , amobarbital (AB, dose: 100 m g / k g ) or diazepam (DZP, dose 5 m g / k g ) through the tail vein or by intraperitoneal injections of carbamazepine (CBZ, dose: 150 m g / k g ) , phenobarbital (PB, dose: 125 m g / k g ) , primidone (PRM, dose: 200 m g / k g ) , or clonazepam (CZP, dose: 200 m g / k g ) . Each group studied was composed of 5 rats. After the injections, the plasma level of each drug was determined serially by the substrate-
0013-4649/90/$03.50 ¢~ 1990 Elsevier Scientific Publishers Ireland, Ltd.
544 labeled fluorescent immuno-assay method in order to identify the maximal drug level (Wong et al. 1979). BAEPs were recorded sequentially, and the peak latency of each wave and the interpeak latency (IPL) between waves I and IV were analyzed.
G. HIROSE ET AL.
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10C Recording of BA EPs BAEPs were recorded sequentially from the 5 rats in each anticonvulsant group 15, 30, 60, 90, 120 and 180 min after injection. Potentials were evoked by a 0.1 msec click given through a speaker placed 40 cm in front of the animals, who were fixed to a board with their heads secured. The click intensity was measured in dB H L relative to the human ear. BAEPs were recorded in response to click stimuli (90 dB, 10/sec) by silver needle electrodes; one inserted subcutaneously at the vertex, and the reference electrode placed behind the right ear after shaving it. As the ground, an additional electrode was inserted into the area just below the left earlobe. The electrode resistances ranged from 3 to 5 kI2. The responses for 2 trials to 1024 click stimuli were averaged and plotted on paper. Averaging was suspended when the animals moved and when there were electrode artifacts. The latencies at the maximal level of each anticonvulsant were measured, and the mean values and their standard deviations calculated. These values were compared statistically with the mean value of each parameter of the BAEPs obtained from 30 similarly anesthetized but untreated rats by the use of Student's t test.
Results The plasma level of each anticonvulsant was measured after injection and the maximal levels were studied. A previous study (Hirose et al. 1986) reported the highest blood level of P H T 15 min after an i.v. injection. We also found the highest blood levels of VPA, ESM and AB 15 min after injection (Fig. 1). The respective maximal blood levels of PB, PRM and CBZ came at 90, 180, and 120 min after injection (Fig. 1).
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Fig. 1. Sequential plasma levels of anticonvulsants after administration, with standard deviations.
Effects of anticonvulsants on latency and IPL Five vertex-positive and 5 vertex-negative peaks were identified in the normal baseline tracings. Positive peaks were labeled from waves I to V. As waves I, II, III and IV were recorded consistently, their latencies and the IPLs between them were studied in each experimental group. From the data obtained, we measured the peak latency of each wave and the IPL. The mean values and standard deviations were also calculated (Tables I and II). When animals were in the toxic state, PHT abolished all the BAEP waves from 5 to 90 min after injection, gradual recovery taking place with the reappearance of each wave (Hirose et al. 1986). PHT is one of the most potent depressants of BAEPs. CBZ significantly delayed the latencies of waves I, II, III and IV. PB significantly delayed the latency of wave IV and CZP that of wave llI (Table I). Representative sequential BAEP changes after CBZ and PB injections are shown in Fig. 2. The other anticonvulsants studied produced no significant changes in the BAEPs even at high dosage. The IPL was not measured for a high
545
EFFECTS OF A N T I C O N V U L S A N T S ON BAEPs TABLE I Latencies of brain-stem auditory evoked potentials after acute intoxication of various anticonvulsants. Blood level (~g/ml)
Peak latencies (msec) I
Control 1.01 _+0,07 Phenytoin 80 m g / k g , i.v. (15 rain) Carbamazepine 150 m g / k g , i.p. (120 rain) Phenobarbital 125 mg/kg, i.p. (90 min) Amobarbital 100 m g / k g , i.v. (15 min) Primidone 200 mg/kg, i.p. (180 min) Ethosuximide 250 mg/kg, i.v. (15 rain) Valproic acid 250 m g / k g , i.v. (15 rain) Clonazepam 200 mg/kg, i.p. (60 rain) Diazepam 5 m g / k g , i.v. (15 n'fin)
II
1II
IV
1,93 +-0,12
2.70 + 0.15
3.68 + 0.16
no waves were elicited
61.3 ± 15.5
1.10+0.04 *
2.15+0.12 *
3.15_+0.17 **
4.31+0.26 **
77.9+32.3
1.02_+0.10
1.98+0.13
2.80-+0.13
3.87+0.15 *
146.6_+47.0
1.07 + 0.08
2.05 -+ 0.22
2.85 _+0.21
3.79 + 0.29
121.2 -+ 13.7
0.99-+0.08
1.93-+0.15
2.69_+0.07
3.66-+0.13
120.9+_43.9
1.01 -+ 0.05
1.87 -+0.06
2.79 -+ 0.09
3.72 +- 0.07
233.7 +- 78.5
0.98 +- 0.05
1.77 _+0.06
2.58 +-0.09
3.50 +-0.11
438.6 +- 30.4
1.06 _+0.06
1.94 +- 0.11
2.90 +- 0.16 *
3.72 _+0.13
0.99 + 0.14
1.88 _+0.14
2.73 _+0.19
3.61 _+0.21
* P < 0.05, * * P < 0.01 (significance levels of differences by Student's t test).
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G. HIROSE ET AL.
546 T A B L E lI Wave I - I V interpeak latency after intoxication with various anticonvulsants. I - I V (msec) Control 2.80 -+ 0.21 Phenytoin 80 m g / k g , i.v. (15 min) Carbamazepine 150 m g / k g , i.p. (120 min) Phenobarbital 125 rng/kg, i.p. (90 rain) Amobarbital 100 m g / k g , i.v. (15 rain) Primidone 200 m g / k g , i.p. (180 min) Ethosuximide 250 m g / k g , i.v. (15 rain) Valproic acid 250 m g / k g , i.v. (15 min) Clonazepam 200 m g / k g , i.p. (60 rain) Diazepam 5 rng/kg, i.v. (15 min)
no waves detected 3.26_+0.31 * * 2.85 +0.16 * 2.72-+0.24 2.68 -+ 0.09 2.71 -+0.10 2.52-+0.09 2.66 _+0.18 2.62_+0.13
* P < 0.05, ** P < 0.01 (significance levels of differences by Student's t test).
dosage of PHT because the BAEPs were abolished; but CBZ and PB significantly prolonged the IPL ( P < 0.01 and P < 0.05) at high dosages (Table
Ii). Discussion
BAEPs have been considered very useful for obtaining information with which to differentiate metabolic from structural diseases of the brainstem. High doses of anesthetics, barbiturates and glutethimide do not alter BAEPs, even when the electroencephalogram is isoelectric (Starr and Achor 1975; Stockard and Sharbrough 1980). Our previous study, however, showed that BAEPs were suppressed in rats treated with a high dose of PHT as well as in a patient suffering acute PHT intoxication (Hirose et al. 1986). In a study of epileptics receiving PHT or PB, or both, chronically, only PHT was associated with prolongation of the IPL at serum levels of more than 20 /~g/ml (Green et al. 1982). Green et al.
concluded that toxic or therapeutic levels of PHT may affect the far-field BAEPs, but they did not mention the possibility of the disappearance of the BAEPs. In our acute experiments, a high dose of PHT given intravenously abolished the BAEPs of rats from 5 to 90 rain after injection, after which the waves reappeared gradually. CBZ did not abolish the BAEP waves at a dose of 150 m g / k g given intraperitoneally, but it did significantly increase each wave latency. Prolongation of the I - I V IPLs was also significant. These data suggest that drugs such as PHT and CBZ may slow central as well as peripheral nerve conduction. Treatment with a high dose of PB prolonged wave IV, and a high dose of CZP wave III, but these prolongations were still within the mean values plus 3 S.D. Under PB intoxication, the I 1V IPL increased significantly but this also was within the mean plus 3 S.D. Whereas barbiturate anesthesia is reported to suppress and abolish auditory evoked potentials (Feldman and Porter 1960), studies done on rats (Bobbin et al. 1979) and humans (Stockard et al. 1977) have shown that their BAEPs are unaffected. In another study, a high dose of pentobarbital given to rats progressively suppressed, then abolished, all the BAEP waves (Shapiro et al. 1984). The dose used in their study was more than 120 m g / k g given intravenously in addition to the initial anesthetic 60 m g / k g given intraperitoneally. Pentobarbital anesthesia was used in our experiments, and its possible synergistic effects might account for the significant latency changes in rats treated with high doses of PHT, CBZ and PB. A dose of 15 m g / k g pentobarbital usually does not affect the latency or amplitude of BAEPs in rats (Bobbin et al. 1979). This dose of pentobarbital, however, was not sufficient to anesthetize our rats and obtain adequate BAEP records; therefore, we used a moderate, 40 m g / k g , dose of pentobarbital. With this dosage, short latency somatosensory evoked potentials have been prolonged in rats (Shaw and Cant 1981). It is therefore possible that the significant changes seen in the animals treated with high doses of CBZ and PB are attributable to synergistic effects produced by pentobarbital anesthesia combined with the anticon-
E F F E C T S OF A N T I C O N V U L S A N T S ON BAEPs
vulsant. We therefore studied the acute effects of anticonvulsants on rat BAEPs to see whether these drugs do, indeed, suppress the amplitude and delay the latencies of some BAEPs waves at maximal serum levels after treatment with non-lethal high doses of anticonvulsants. We compared the peak BAEP latencies at the maximal anticonvulsant levels with those at the control level of the anesthetic pentobarbital. During sequential recording of the BAEPs, in spite of the decreased effect of the anesthesia after intraperitoneal injection, for each BAEP peak latency there was more delay as time passed (Fig. 2). These findings are evidence of the acute effects of anticonvulsants on BAEPs. Starr and his associates reported normal latencies and amplitudes for all the BAEP components in overdoses of barbiturates, diazepam, glutethimide, amitriptyline, imipramine, propoxyphene and perphenazine (Starr and Achor 1975). Their series did not include overdoses of PHT and CBZ. We postulate that patients treated with high doses of anticonvulsants may show neurophysiological changes in their BAEPs. Because of insufficient data for comparative pharmacokinetics, we cannot translate our data on the toxic levels of each anticonvulsant from rats to human patients. But, if the effects on human BAEPs are similar to those found for rats, BAEPs will have to be interpreted very carefully. Abolition of all BAEP waves after wave I for patients in whom brain-stem reflexes are absent is often used as a criterion of brain death (Goldie et al. 1981). Our data show that PHT abolished all the BAEP waves in toxic rats, that this phenomenon was completely reversible and that CBZ and PB produced significant delays of latencies and prolongation of the IPL between waves I and IV. We conclude that loss of BAEPs is consistent with brain death only when PHT intoxication has been excluded. Cases of delayed BAEP latencies
547
must be interpreted very carefully, taking into account the possibility that such changes may be caused by anticonvulsants such as CBZ and PB. We thank Miss Hiroko O k u m u r a for her invaluable secretarial assistance.
References Bobbin, R.P., May, J.G. and Lemoine, R . L Effects of pentobarbital and ketamine on brainstem auditory potentials. Latency and amplitude intensity functions after intraperitoneal administration. Arch. Otolaryngol., 1979, 105: 467-470. Feldman, S. and Porter, R.W. Long latency responses evoked in the anterior brain stem under pentobarbital anesthesia. Electroenceph. clin. Neurophysiol., 1960, 12:111 118. Goldie, W.D., Chiappa, K.H., Young, R.P. and Brooks, E.B. Brainstem auditory and short-latency somatosensory evoked responses in brain death. Neurology, 1981, 31: 248-256. Green, J.B., Walcoff, M.R. and Lucke, J.F. Comparison of phenytoin and phenobarbital effects on far-field auditory and somatosensory evoked potential interpeak latencies. Epilepsia, 1982, 2 3 : 4 1 7 421. Hirose, G., Kitagawa, Y., Chujo, T., Oda, R., Kataoka, S. and Takado, M. Acute effects of phenytoin on brainstem auditory evoked potentials: clinical and experimental study. Neurology, 1986, 36: 1521-1524. Shapiro, S.M., Moiler, A.R. and Shiu, G.K. Brain-stem auditory evoked potentials in rats with high-dose pentobarbital. Electroenceph. clin. Neurophysiol., 1984, 58: 266-276. Shaw, N.A. and Cant, B.R. The effect of pentobarbital on central somatosensory conduction time in rat. Electroenceph. clin. Neurophysiol., 1981. 5 1 : 6 7 4 677. Starr, A. and Achor, L.J. Auditory brain stem responses in neurological disease. Arch. Neurol., 1975, 32:761 768. Stockard, J.J. and Sharbrough, F.W. Unique contributions of short latency auditory and somatosensory evoked potentials to neurologic diagnosis. Prog. Clin. Neurophysiol.. 1980, 7: 23l 263. Stockard, J.J., Rossiter, V.S., Jones, T.A. and Sharbrough, F.W. Effects of centrally acting drugs on brain stem auditory responses. Electroenceph. clin. Neurophysiol., 1977, 43: 550-551. Wong, R.C., Burd, J.F., Carrico, R.J., Buckler, R T . , Thomas, J. and Boguslaski, R.C. Substrate-labeled fluorescent immunoassay for phenytoin in h u m a n serum. Clin. Chem., 1979, 25: 686-691.