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Dextromethorphan-Facilitated
Anticonvulsant Effects Of Electroacupuncture In
Rat Electroconvulsive Shock (ECS) Model
Huanmin Gao, MD
Yaoquan Liu, MD
Shaoping Wang, MD
Shaoping Lu, MD
Bo Xiang, MD
ABSTRACT
Background Our previous study demonstrated that electroacupuncture (EA) is beneficial to attenuate seizure induced by electroconvulsive shock (ECS).
Objective To investigate whether dextromethorphan augments the antiepileptiform effects of EA in rats.
Design and Setting We studied 65 male Wistar rats weighing 280-300 g. The rats were randomized to 8 groups: normal control group (n=5), seizure group (n=7), EA (n=9), sham EA (n=7), drug vehicle (n=8), dextromethorphan (n=8), EA + vehicle (n=10), and EA + dextromethorphan (n=11). The seizure model induced by repeat ECS (500 V, 60 mA, 50-Hz sine waves, clipped on both ears 3 times in 5-minute intervals) was used. EA was given between the acupuncture points Fengfu point (GV 16) and Jinshuo point (GV 8), with dense-sparse waveforms, which can be transformed into each other when a dense or sparse wave is terminated.
Main Outcome Measure Ictal behavior and EEG epileptiform readings in each group.
Results After repeated ECS, the ictal behavioral and electroencephalogram (EEG) epileptiform was induced in all cases. EA significantly suppressed the ictal behavioral and EEG epileptiform. Intracerebral microinjection of dextromethorphan (5 mg/0.5 mL) partially blocked the ictal behavioral and EEG epileptiform. EA, given 15 minutes after dextromethorphan administration, enforced the suppression of EEG compared with EA+vehicle group or EA group (P<.05).
Conclusions These results implied that EA is effective to attenuate seizures induced by ECS, and dextromethorphan facilitated the anticonvulsant effects of EA in rats.
KEY WORDS
Electroacupuncture, Electroconvulsive Shock, Seizure, Dextromethorphan
INTRODUCTION
Acupuncture may be effective in treating epilepsy to some extent, shortening the duration of convulsion and attenuating the seizure.1-4 Many efforts have been made to search medical literature in understanding the effects of acupuncture manually or electrically.1,4,5 Among the relevant agents, dextromethorphan is a potent neuroprotectant and anticonvulsant.6 Dextrophan, the major metabolite of dextromethorphan, used intracerebrally pretreatment, significantly attenuated seizures induced by kainic acid in a dose-related manner.6,7 Because of relatively few adverse effects, dextromethorphan is available as an over-the-counter drug in many countries. However, whether dextromethorphan improves the effects of electroacupuncture (EA) and ameliorates the neuronal damages is not known. Therefore, our study had 2 aims: to investigate whether EA is beneficial to attenuate seizure induced by electroconvulsive shock (ECS) and to examine if dextromethorphan enhances the anticonvulsant effects of EA in rats.
METHODS
ECS Seizure Model
The ECS seizure model in rats has been described previously.4,5
The Animal Subjects Committee of Qingdao University approved this protocol. The animals were treated humanely and our study conformed to the standards of current ethical animal research practices. We studied 65 male Wistar rats weighing 280-300 g. The rats were randomized to 8 groups: normal control group (n=5), seizure group (n=7), EA (n=9), sham EA (n=7), drug vehicle (n=8), dextromethorphan (n=8), EA + vehicle (n=10), and EA + dextromethorphan (n=11).
Rectal temperature was continuously monitored and maintained at 37°C with an electric warming pad. Room temperature was kept at 25°C by air conditioning.
The animals were confined with a stereotactic apparatus (SN-3, Narishige, Tokyo, Japan). Both ears were clipped to the apparatus generating 50-Hz sine wave (500 V, 60 mA, duration: 400 ms, 3 times at 5-minute intervals).
Ictal behavioral and EEG monitoring confirmed this seizure model. Elastic gauge sensors were touched at the breast and the right upper limb, respectively. The sensors were linked to biosensor amplifier (t0.3), then to the electric integrator, and finally stored into the computer for processing. The movement amount analysis software (MAAS 1.2 for rat, version 1.2) was obtained from the Department of Physiology, Qingdao Medical College. Data sampling periods were 5 minutes each. The total amount of breath and limb movement was present as mV 2 xseconds. Movement (limb or thorax) amount was presented as percentage of baseline (i.e., stale seizure stage).
Intracerebral Microinjection
Anesthesia was induced by 8% chloralhydrate (150 mg/kg, ip). The stainless cannula (outer diameter 0.75 mm, inner diameter 0.4 mm) was implanted into the right cerebroventricule stereotactically. The tip of the cannula was at A 1.0 mm, R 1.0 mm, H 4.2 mm according to the atlas of rat brain. A total of 0.5 mL of ACSF (artificial cerebrospinal fluid) as vehicle or drug was injected within 5 minutes.
Electroacupuncture Application
EA was applied at the acupuncture points of Fengfu point (GV 16) and Jinshuo point (GV 8) according to Traditional Chinese Medicine (TCM), similar to that in humans but using a depth 2 mm subcutaneously (using model G6805-2 EA apparatus; Shanghai Medical Electronic Apparatus Company, China) (Figure 1). EA started 15 minutes after microinjection and lasted for 1 hour. EA consisted of dense-sparse waveforms, which can be transformed into each other when a dense or sparse wave is terminated. The stimulating parameters of dense wave were frequency 18 Hz, intensity 1.2~1.4 mA, duration 1.05 seconds; those of the sparse waves were frequency 3.85 Hz, intensity 1.0~1.2 mA, duration 2.85 seconds.
EA Control
Acupuncture control treatments have been used as placebo controls. EA control in animals cannot precisely simulate true acupuncture. Acupuncture was performed in the traditional way rather than on certain fixed acupoints only with no teh-Qi ("obtained essence," according to TCM), from qualified acupuncturists. In this study also, the power to the EA apparatus was off.
Electroencephalographic
(EEG) Recording and Power Spectral Analysis
Needle electrodes (Ag-Cl) were inserted bilaterally into the scalp at the sensorimotor area A3, L3, which related to seizure and the EEG signal was easy to record. EEG recordings were stored on the computer for frequency analysis. Fourier transformation was carried out with the computer. The parameters were time constant: t0.2, high frequency cut: 100 Hz, sensitivity 0.05 mV/cm, sampling time 40 seconds, sampling frequency: 200 Hz, amplifying 10, frequency range: 1-30 Hz. Relative unit of EEG power spectrum was used. Increased amplitude and frequency of EEG can be digitally indicated as an increase of EEG power in the relative unit.
Brain Histopathology
Seven days after the experiment and under 8% chloralhydrate (300 mg/kg, i.p.) anesthesia, the rat brain was perfused with 0.9% normal saline, then 4% paraformaldehyde via the left ventricle, with the descending aorta clipped. Rats were killed humanely. The brain was carefully removed and immersed into the 4% buffered paraformaldehyde+20% sucrose for 2 days at 4°C, then transferred into 30% sucrose perfusion fixative for 2 days at 4°C.
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Figure 1. Schematic diagram showing the acupuncture points in rat and waveforms of electrical pulse while using electroacupuncture apparatus.
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Figure 2. Time courses of ECS-induced seizure in rats.
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The brain was then serially sectioned in a coronal plane into 30-micron sections with frozen microtome (Jiangwan, Shanghai, China). Sections were sequentially identified and stained with hematoxylin-eosin.
According to the rat brain atlas, the brain slice at A4.0 section was scanned using image-processing system (Leica Q500 IW, Germany).
Three view fields in CA1 pyramidal cell were selected for the average. The cell density was measured. The residual cell density ratio was calculated as percentage of the cell density ipsilaterally/contralaterally.
Statistical Analysis
We used SPSS 11.5 for Windows for statistical analysis (SPSS Inc, Chicago, IL). Differences in the EEG power spectrum before and after treatments or between groups were tested by repeated-measures analysis of variance (MANOVA). Cell density was examined with intergroup comparisons. An a level of .05 was used to determine statistical significance; all statistical testing was 2-sided. Values are presented as mean (SD).
RESULTS
Seizure Induced By Electroconvulsive Shock Treatment
Repeated ECS produced a characteristic seizure that was confirmed by EEG epileptiform and ictal behavior. The EEG monitoring showed a heterogeneous pattern of electrophysiological abnormalities. The ictal behavior included increased limb activities and respiratory movements as well as biting, tearing, and salivary secretions. The consistent observation was increase of EEG amplitude and appearance of spike and slow waves including u, d after repeated ECS, which affected both hemispheres.
The t time was from 0.5 hours to 7 hours; the stable period was 1-5 hours in all cases (Figure 2). In the previous study,4 58% of cases still had EEG epileptiform discharge at 3 days following ECS stimulations and the mortality rate was 10% at 7 days, which was much higher than that of the normal group. Data presented in Figure 2 show that the ECS-induced seizure-like phenomena were stable.
Our results demonstrated that the upper and lower extremity movements and breath frequency increased, respectively; in most cases, tears and saliva dripped out. The quantitative analysis of the limb and breast respiration showed that the integrated activities per time unit (5 minutes) increased significantly compared with the control group, or compared within this group before ECS treatment (P<.05 for each). Spike and slow waves of EEG appeared frequently in all seizure cases. EEG total power increased significantly (P<.05) compared with the control group (Figure 2).
Therefore, in this model, the stable seizure was long enough to offer a platform for further research. The results of EEG and animal behavioral changes implied that repeated ECS could produce a stable model of a seizure in the rat.
Effects of Dextromethorphan on Seizure
According to our pilot study on the dosage of dextromethorphan on this seizure model, the dose for intracerebral microinjection was determined at 5 mg/ 0.5 mL. In this study, significantly decreased EEG amplitude and number of spikes per minute also were found 30 minutes after microinjection of dextromethorphan (5 mg/0.5 mL) compared with the control group (P<.05) (Table 1).
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Table 1. Changes After Dextromethorphan
Intrahippocampal Microinjection
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Mean (SD)
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P Value vs Vehicle
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Control
(n=5)
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Vehicle
(n=8)
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Dextromethorphan
(n=8)
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Spikes per
minute
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0.00
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47.50
(12.30)
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28.30
(8.30)
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<.05
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EEG total power
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1.02
(0.12)
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2.83
(0.12)
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1.78
(0.18)
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<.01
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Limb movement
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1.07
(0.21)
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3.89
(0.22)
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2.87
(0.28)
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<.01
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Respiration
movement
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1.03
(0.11)
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2.82
(0.24)
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1.67
(0.19)
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<.01
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% of cell density
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99.50
(4.62)
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60.30
(7.91)
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75.20
(9.40)
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<.05
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Abbreviation: EEG, electroencephalogram.
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Table 2. Effects of Electroacupuncture (EA)
on Seizures Induced by ECS in Rats*
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Mean (SD)
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EA Control (n=7)
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EA (n=9)
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P Value
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Spikes per minute
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48.5 (11.3)
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29.3 (9.3)
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<.05
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EEG total power
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2.81 (0.16)
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1.67 (0.14)
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<.01
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Limb movement
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3.79 (0.25)
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2.78 (0.58)
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<.01
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Respiration movement
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2.88 (0.27)
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1.77 (0.39)
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<.01
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% of cell density
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62.3 (7.11)
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76.2 (9.9)
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<.05
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*EA started 75 minutes after electroconvulsive shock (ECS) and lasted for 1 hour.
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Table 3. Effects of Dextromethorphan on Electroacupuncture (EA) Anticonvulsion in Rat Electroconvulsive Shock Model*
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Mean (SD)
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EA + Vehicle
(n=10)
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EA +
Dextromethorphan (n=11)
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Spikes per minute
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25.5 (5.3)
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16.8 (8.3)
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EEG total power
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1.68 (0.36)
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1.47 (0.24)
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Limb movement
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1.79 (0.35)
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1.48 (0.51)
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Respiration movement
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1.87 (0.37)
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1.37 (0.29)
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% of cell density
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74.3 (7.91)
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79.2 (9.6)
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*All comparisons P<.05 between groups. EA started 15 minutes after microinjection of 0.5 mL of drug or vehicle and lasted for 1 hour.
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Under the microscope, most of the neurons were eumorphism with the clear nucleus at the cortex, striatum, and hippocampus. The CA1 and CA3 of the hippocampus showed normal cytoarchitecture. Neuron loss was apparent in all cases with seizure except for the control group; the residual neuron had incomplete cellular structure. Cluster or scatter necrotic neurons could be seen in the hippocampus. The pyramidal cell density was increased significantly in the dextromethorphan group (P<.05) compared with the drug vehicle group.
Dextromethorphan exerts potent antiepileptiform effects that are characteristic for NMDA receptor antagonism, as described by Loscher and Honack.7 Our study showed that dextromethorphan had both antiepileptiform effects and a neuroprotective effect in the ECS rat model.
Anticonvulsant Effects of EA
In our study, EA had the anticonvulsant effects on rats' seizure model induced by repeated ECS. The numbers of spikes per minute began to decrease 25 minutes after EA therapy and reached a stable level at 40 minutes. The amplitude of EEG began to decrease 15 minutes after EA therapy and reached a stable level at 30 minutes. The epileptiform activities were partially concurred by EA treatment. EA accelerated the recovery of EEG readings. The rats showed less ictal activities including less tearing and salivary secretions. The changes of EEG after EA therapy are shown in Table 2.
Dextromethorphan Facilitated the Anticonvulsant Effects of EA
Compared with EA plus vehicle as the control group, EA + dextromethorphan decreased the spike frequency and the amplitude, the total EEG power, and the numbers of spikes per minute significantly (P<.05) (Table 3). However, the pyramidal cell density in the CA1 region of hippocampus at section 4 merely showed the tendency of increasing (from mean [SD] 74.3% [7.91%] to 79.2% [9.6%]), but not significantly. This may be attributed to the limited sample size.
From the comparison of EA + vehicle group and EA plus dextromethorphan group, the effects of dextromethorphan may therefore be obvious: it facilitated the anticonvulsant effects of EA by decreasing the epileptiform waves in this model.
DISCUSSION
Clinical and laboratory studies have shown that acupuncture is an effective method in the treatment of epilepsy.1-5 However, acupuncture cannot totally control seizures. Therefore, many efforts have been made to select some augmentation of the effects of acupuncture. In our previous study, an agonist of kappa opioid receptors, microinjected into the hippocampus, inhibited seizure by electroacupuncture in rats.4
Known antagonists of the NMDA receptor-channel complex may be useful for the treatment of thrombotic stroke, head injury, and epilepsy. Their clinical use, however, could be limited by the incidence of adverse effects such as ataxia. Dextrophan, the major metabolite of dextromethorphan, attenuated seizures induced by kainic acid and exerted anticonvulsant effects, which are characteristic for NMDA receptor antagonism.8 NMDA antagonists as anticonvulsants are especially active in preventing the generalization of the behavioral and electrical seizures, and display a typical spectrum of in vitro antiepileptiform activities.9 As neuroprotective drugs, NMDA antagonists are effective against many types of neuronal injury and show a 1-2 hour window of activity, suggesting an influence of NMDA receptors in the early or acute mechanisms of brain damage. Dextromethorphan , an over-the-counter drug, appears to be a potent neuroprotectant as well as anticonvulsant.10
Hence, in this study, we chose dextromethorphan at the micromolar level, which acts on NMDA receptors, rather than at the nanomolar level, which acts at sigma receptors selectively.
We may have proven that acupuncture has the effects of ameliorating the brain blood supply and altering the blood viscosity.11,12
Thus, EA may have neuroprotective effects in stroke, head injury, and epilepsy.13 EEG is an often-used indicator to reflect the functional status of the brain. The effects of dextrophan and dextromethorphan were tested using the electroencephalogram (EEG) and behavioral effects induced by topical cortical application of penicillin in rabbits.14 In this seizure model, seizure induced by repeated ECS was reflected by a heterogeneous pattern of electrophysiological abnormalities. Our consistent observation was an increase of EEG amplitude and appearance of spike and slow waves including u, d in both hemispheres. After EA treatment, the numbers of spikes per minute began to decrease and reached a stable level at 40 minutes. The amplitude of EEG began to decrease 15 minutes after EA therapy and reached a stable level at 30 minutes. The epileptiform activities were partially controlled by EA treatment. EA accelerated the recovery of EEG.
The neuropathological study showed that cluster or scatter necrotic neurons could be seen in the hippocampus of the seizure rats. EA could significantly increase the pyramidal cell density. As a result, it may be inferred that EA has a neuroprotective as well as anticonvulsant effect in this seizure model.
CONCLUSIONS
Our results suggest that repeated ECS produced a stable seizure in rats. EA has anticonvulsant effects and dextromethorphan facilitated the anticonvulsant effects of EA in rats.
Neuroprotectants such as dextrophan may augment the effects of EA anticonvulsion, providing the possibility of combining the use of acupuncture and dextromethorphan in the treatment of epilepsy.
FUNDING/SUPPORT
The present work is supported by The Research Foundation for M.D. Physicians of Qingdao Central Hospital, No. 04B01A, in China.
REFERENCES
- Wang BE, Yang R, Cheng JS. Effect of electroacupuncture on the level of preproenkephalin mRNA in rat during penicillin-induced epilepsy. Acupunct Electrother Res. 1994;19(2-3):129-140.
- Lewis C, Halvorsen R. Training in acupuncture. BMJ. 2003;326:S152.
- Shen D. Review of epilepsy treatment by acupuncture. J Jiangsu Tradit Chin Med. 1986;7(12):18-20.
- Gao H, Cheng J. Role of intrahippocampal kappa opioid receptors in inhibiting the seizure by electroacupuncture (EA) in rats. Acupuncture Res. 1998;23(2):122-125.
- He X, Cao X. Effects of intrahippocampal delta receptors on inhibition of electroconvulsive shock by electroacupuncture. Acta Pharmacol Sinca. 1989;10(3):197.
- Kim HC, Bing G, Jhoo WK, et al. Metabolism to dextrorphan is not essential for dextromethorphan's anticonvulsant activity against kainate in mice. Life Sci. 2003;72(7):769-783.
- Loscher W, Honack D. Differences in anticonvulsant potency and adverse effects between dextromethorphan and dextrorphan in amygdala-kindled and non-kindled rats. Eur J Pharmacol. 1993;238(2-3):191-200.
- Carter AJ. Many agents that antagonize the NMDA receptor-channel complex in vivo also cause disturbances of motor coordination. J Pharmacol Exp Ther. 1994;269(2):573-580.
- Zhang Cl, Gloveli T, Heinemann U. Effect of NMDA and AMPA receptor antagonists on different forms of epileptiform activity in rat temporal cortex slices. Epilepsia. 1994;35(suppl 5):S68-S73.
- Sagratella S. NMDA antagonists: antiepileptic-neuroprotective drugs with diversified neuropharmacological profiles. Pharmacol Res. 1995;32(1-2):1-13.
- Omura Y. Normal and abnormal relationship between brain circulation estimated by supraorbital temperature measurements and grasping force of the corresponding hands: their clinical application for diagnosis and evaluation of various modes of treatment. Acupunct Electrother Res. 1978;3(1-2):49-96.
- Gao H, Guo J, Zhao P,et al. The neuroprotective effects of electroacupuncture on focal transient cerebral ischemia in monkey. Acupunct Electrother Res. 2002;27(1):45-57.
- Wang JX, Liu F, Xu YM, et al. Clinical and hemarrhyological observation on the treatment of cerebral infarction with acupuncture plus collateral needling. Chin Acupuncture Moxibustion. 1991;3:1-4.
- Zeng YC, Pezzola A, Scotti De Carolis A, et al. Inhibitory influence of morphinans on ictal and interictal EEG changes induced by cortical application of penicillin in rabbits: a comparative study with NMDA antagonists and pentobarbitone. Pharmacol Biochem Behav. 1992;43(2):651-656.
AUTHORS' INFORMATION
Dr Huanmin Gao is Associate Professor of Neurology, Department of Neurology, The Second Affiliated Hospital of Qingdao University Medical College, in P.R. China.
Huanmin Gao, MD, PhD*
Associate Professor, Department of Neurology
The Second Affiliated Hospital of Qingdao University Medical College
127 Si Liu South Rd
Qingdao 266042
P.R. China
Phone: +86-532-84961748 • Fax: +86-532-84863506
E-mail: huanmgao@yahoo.com.cn
Dr Yaoquan Liu is Associate Professor of Neurology, Dept of Neurology, at The Second Affiliated Hospital of Qingdao University Medical College, in P.R. China.
Yaoquan Liu, MD, MS
The Second Affiliated Hospital of Qingdao University Medical College
127 Si Liu South Rd
Qingdao 266042
P.R. China
Phone: +86-532-84961748 • Fax: +86-532-84863506
Dr Shaoping Wang is Associate Professor of Neurology, Dept of Neurology, The Second Affiliated Hospital of Qingdao University Medical College, in P.R. China.
Shaoping Wang, MD, MS
The Second Affiliated Hospital of Qingdao University Medical College
127 Si Liu South Rd
Qingdao 266042
P.R. China
Phone: +86-532-84961748 • Fax: +86-532-84863506
Dr Shaoping Lu is Attending Physician of Neurology at The Second Affiliated Hospital of Qingdao University Medical College, in P.R. China.
Shaoping Lu, MD, MS
The Second Affiliated Hospital of Qingdao University Medical College
127 Si Liu South Rd
Qingdao 266042
P.R. China
Phone: +86-532-84961748 • Fax: +86-532-84863506
Dr Bo Xiang is a Resident of Neurology at The Second Affiliated Hospital of Qingdao University Medical College, in P.R. China.
Bo Xiang, MD, MS
The Second Affiliated Hospital of Qingdao University Medical College
127 Si Liu South Rd
Qingdao 266042
P.R. China
Phone: +86-532-84961748 • Fax: +86-532-84863506
*Correspondence and reprint requests
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