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Vinpocetine prevents 4-aminopyridine-induced changes in the EEG, the auditory brainstem responses and hearing
Clinical Neurophysiology 115 (2004) 2711–2717 www.elsevier.com/locate/clinph
Vinpocetine prevents 4-aminopyridine-induced changes in the EEG, the auditory brainstem responses and hearing Maria Sitgesa,b,*, V. Nekrassova,b a
Depto. de Biologı´a Celular y Fisiologı´a, Instituto de Investigaciones Biome´dicas, UNAM, Apartado Postal 70228, Ciudad Universitaria 04510, Mexico City, DF, Mexico b Instituto Nacional de la Comunicacion Humana, CNR, SSA, Mexico, DF, Mexico Accepted 28 June 2004
Abstract Objective: The purpose of the present study was to investigate if the sodium channel blocker and memory enhancer, vinpocetine, was capable to overcome the epileptic cortical activity, the abnormalities in the later waves of the auditory brainstem responses (ABRs) and the hearing loss induced by 4-AP at a convulsing dose in the guinea pig in vivo. Methods: EEG and ABR recordings before and at specific times within 2 h after the injection of 4-AP (2 mg/kg, i.p.) were taken in animals pre-injected i.p. with vehicle or with vinpocetine (2 mg/kg) 1 h before 4-AP. The amplitude and latency of the ABR waves induced by a monoaural stimulus of high intensity (100 dB nHL) at 4 and 8 kHz pure tone frequencies and the ABR threshold were determined in the animals exposed to the different experimental conditions. Results: Vinpocetine inhibited the EEG changes induced by 4-AP for the ictal and post-ictal periods as well as the alterations in amplitude and latency of P3 and P4 and the increase in the ABR threshold induced by 4-AP. Conclusions: Vinpocetine prevents the retro-cochlear alterations and the hearing decline that accompany the epileptic cortical activity. Significance: Vinpocetine could be a promising alternative for the treatment of epilepsy. q 2004 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Antiepileptic drugs; Cognitive functions; Epilepsy; Lateral superior olive; Medial superior olive; Audition
1. Introduction Auditory brainstem responses (ABRs) are far fieldevoked potentials that consist of several waves that occur within 10 ms post-stimulus. In patients ABRs are useful in the clinical diagnosis of retro-cochlear lesions, because abnormalities of the later wave components of the ABRs are indicative of alterations in their generators that are localised in specific nuclei of the auditory brainstem (Hughes and Fino, 1985). Determination of the ABRs threshold is also an objective measure of the hearing sensitivity; because while * Corresponding author. Address: Depto. de Biologı´a Celular y Fisiologı´a, Instituto de Investigaciones Biome´dicas, UNAM, Apartado Postal 70228, Ciudad Universitaria 04510, Mexico City, DF, Mexico. Tel.: C525-5622-3866; fax: C525-5622-3897. E-mail address: [email protected] (M. Sitges).
the hearing sensitivity declines, stimuli of progressively higher intensity (in dB) are needed for evoking the ABRs. The epileptic cortical activity induced in the guinea pig by the convulsing agent 4-aminopyridine (4-AP) is accompanied by specific alterations of the later waves of the ABRs, that resulted in hearing decline (Nekrassov and Sitges, 2003). Glutamate is the most important excitatory neurotransmitter in the brain. Therefore, epilepsy can be induced by increasing the cerebral excitatory neurotransmission with drugs such as 4-AP, that in vivo has shown to increase particularly glutamate release (Morales-Villagran and Tapia, 1996). Many of the most widely used antiepileptic drugs suppress the abnormal neuronal excitability associated with seizures by means of complex voltage- and frequency-dependent inhibition of ionic currents through sodium channels (Lingamaneni and Hemmings, 2003;
1388-2457/$30.00 q 2004 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2004.06.019
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Ragsdale and Avoli, 1998). Vinpocetine (ethyl apovincamine-22-oate) is a sodium channel blocker (Erdo¨ et al., 1996), that selectively inhibits neurotransmitter release triggered by increased sodium channel permeability (Sitges and Nekrassov, 1999; Trejo et al., 2001). For instance, the release of glutamate induced by the activation of sodium channels with veratridine in cerebral isolated nerve endings is inhibited by vinpocetine (Sitges and Nekrassov, 1999), as well as by carbamazepine and other antiepileptic drugs (Ambrosio et al., 2000). In the guinea pig in vivo vinpocetine inhibits the alterations of some of the ABR waves and the hearing loss induced by the aminoglycoside antibiotic, amikacin (Nekrassov and Sitges, 2000). Also previous studies have shown that vinpocetine improves cognitive functions in rats (DeNoble, 1987; DeNoble et al., 1986), as in healthy human volunteers (Bhatti and Hindmarch, 1987; Subhan and Hindmarch, 1985). An elegant compilation of the basic and clinical pharmacology of the nootropic drug vinpocetine can be found in Bo¨no¨czk et al. (2000). Since one major problem of epilepsy is the deleterious cognitive consequences that, among other factors, are caused by the illness (Jokeit and Ebner, 1999; Meador, 2001; Prevey et al., 1998; Theodore et al., 1999), in the present study the antiepileptic potential of vinpocetine was explored by testing its effect on the epileptic cortical activity, the alterations on the later ABR waves and the hearing decline induced by the convulsing agent 4-AP in the guinea pig in vivo.
2. Methods Experiments were performed in pigmented adult male guinea pigs initially weighing 312G17 g. EEG recordings were used to evaluate changes in cortical excitability and ABR recordings to evaluate hearing. For EEG recordings, needle electrodes were placed subcutaneous over the left temporal area (active electrode) and over the left frontal area between the midline and the arched portion of the orbital crest (reference electrode). For ABR recordings, needle electrodes were placed subcutaneous at the ipsilateral left pinna (reference electrode), the contralateral pinna (ground electrode) and the vertex (active electrode). EEG and ABR recordings were performed in a sound proof room with a Nihon-Kohden Neuropack IV Mini (MEB-5304 K) system. Prior to each sequence of recordings, guinea pigs were anaesthetised with ketamine (50 mg/kg/10 mg/kg xylazine, i.p.) for restraining movement, stress and muscular activity. This anaesthetic was chosen because in the guinea pig it does not change the latency or the amplitude of the ABR waves or the ABR threshold within the time of the experiments (Nekrassov and Sitges, 2003). Recordings were taken following methods previously reported (Nekrassov and Sitges, 2000, 2003). For the ABR recordings monaural tone burst stimuli of 4 and 8 kHz were
delivered by TDH 39 earphone located 1 cm from the left ear. The right ear was blocked with a special wax plug that substantially reduced the sound level at this ear. Four or 8 kHz alternating polarity tone bursts (20/s), with 2 ms duration and 0.5 ms rise-fall times were used for evoking the potentials. Responses were amplified and averaged (500 responses), displayed vertex positive up and saved to disk for off-line analyses. The Institutional Animal Use and Care Committee approved all experimental procedures. 2.1. Animals and treatments 4-AP, obtained from Sigma (St Louis MO), was dissolved in oxygenated HEPES buffer (composition in mM: 127 NaCl, 1.18 KH2PO4, 3.73 KCl, 1 CaCl2, 1.18 MgSO4, 20 HEPES and 5.6 mM dextrose). The dose of 4-AP was chosen on the basis of preliminary experiments in non-anaesthetised animals injected with several doses (in mg/kg: 1, 2, 4 and 6) of 4-AP. In the guinea pig, the dose of 2 mg/kg 4-AP was chosen to carry out the experimental series of the present study because the tonic–clonic convulsions induced by this dose that appear about 20 min after the injection of 4-AP last for about 10 min and the animals recuperate few hours after the end of the experiments. In contrast, in the animals (seven) injected with 4 mg/kg 4-AP the duration of the convulsions lasted for more than 1 h, and all the animals (four) injected with 6 mg/kg 4-AP died in status epilepticus. In the animals injected with 1 mg/kg the tonic–clonic convulsions were rare. Vinpocetine (kind gift of Armstrong International) was dissolved in saline acidified with HCl and adjusted to pH 4 with NaOH. The dose of vinpocetine was chosen also on the basis of preliminary experiments. For instance, the effect of vinpocetine at increasing concentrations (range from 0.5 to 5 mg/kg) was first tested on the tonic–clonic convulsions induced by the injection of 2 mg/kg 4-AP in anaesthetised animals. Since from the dose of 2 mg/kg, the anticonvulsant effect of vinpocetine was clearly observed, this dose of vinpocetine was chosen to carry out the experiments in the anaesthetised animals. Vinpocetine was injected 1 h before 4-AP because when injected longer before (3–4 h) its inhibitory effect was less pronounced in some animals. In other preliminary experiments the effect of 5 mg/kg vinpocetine injected 1 h before a higher dose (4 mg/kg) of 4-AP was tested. Under this experimental conditions only in half of the animals vinpocetine clearly inhibited the severe epileptic cortical activity induced by the convulsing agent at this high dose. Five guinea pigs were used for the experimental series presented in this study. In a first set of experiments these animals were injected with vehicle 1 h before anaesthesia. Vehicle refers to the solution used to dissolve vinpocetine. For testing the effect of 2 mg/kg vinpocetine, a second set of experiments in which the same animals were injected with vinpocetine 1 h before anaesthesia was done 10 days after
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the first set of experiments. Ten minutes after the injection of the anaesthetic solution 3 types of recordings: (1) the ABR recordings elicited by a stimulus of high intensity (100 dB), (2) the ABR recordings for determination of the auditory threshold and (3) the EEG recordings, were obtained in the animals pre-injected with vehicle and, 10 days after, in the same animals pre-injected with vinpocetine. Immediately after getting those recordings, the animals were injected (i.p.) with 4-AP 2 mg/kg for obtaining a new series of recordings taken at specific times after 4-AP injection. For instance, the EEG recordings for the ictal period were taken about 20 min after the injection of 4-AP, and the ABR recordings evoked by 100 dB or the EEG recordings for the post-ictal period were taken 30, 60, 80 and 100 min after the injection of 4-AP. The recordings for determination of the ABR thresholds were taken 30 and 60 min after 4-AP. All the recordings were taken within the first 2 h after 4-AP injection taking into account the start and duration time of the ictal and post-ictal periods induced by the convulsing agent. 2.2. Determination of ABR wave parameters The latency and amplitude of each wave component of the ABR elicited by the stimulus of high intensity (100 dB) with pure tone frequencies of 4 or 8 kHz was measured in all the ABR recordings obtained under the different experimental conditions at the specified times. The latency of each ABR wave (in ms) refers to the time interval between the onset of the auditory stimulus and the positive peak of the wave. The onset of the stimulus is indicated by the vertical arrow at the bottom of the recordings on Fig. 4. The peak amplitude (in mV) of each wave of the ABR is the difference between the positive peak of the wave and the reference baseline (trace between the stimulus and the appearance of the first wave of the ABRs on Fig. 4). 2.3. Determination of the hearing sensitivity ABR recordings elicited by stimuli of progressively lower intensity (in dB) were taken for determining the hearing threshold in the animals exposed to the different experimental conditions. Threshold is defined as the lowest stimulus intensity (in dB) at which the P3 wave of the ABR could still be recorded in three consecutive trials (each trial equals the average response to 500 stimuli). For obtaining the auditory threshold a tone burst response induced with an intensity of 100 dB normal hearing level (nHL) was initially recorded. Then thresholds were determined for each stimulus by reducing the intensity at 20 and 10 dB nHL intervals, and then at 5 dB intervals down from a suprathreshold ABR recording to identify the lowest level at which reproducible waves could be recognised. The sound threshold level was also objectively estimated. The amplitude of the P3 wave at the peak was plotted against the corresponding sound intensity in order to construct
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input–output curves at 20, 10 and 5 dB nHL steps for 4 and 8 kHz. 2.4. Statistics The differences between results obtained before and at specific times after 4-AP and the differences between results obtained in the absence (vehicle) and presence of vinpocetine were considered statistically significant when P was less than 0.05 using a repeated measure analysis of variance and a post-hoc Tukey’s Studentised test. All data are expressed as meansGstandard error of the mean.
3. Results 3.1. Vinpocetine inhibits all the changes induced by 4-AP on the EEG All anaesthetised animals injected with 2 mg/kg 4-AP developed generalised seizures. The onset of seizure activity, characterised by repetitive high amplitude spikesharp wave activity in the EEG tracing appears about 20 min after the injection of 4-AP. This change on the cortical activity elicited by 4-AP during convulsions is followed by a pattern of cortical activity characterised by isolated spikes of higher amplitude. The time of duration of this later pattern of cortical activity (not accompanied by convulsions) is referred to as the post-ictal period. The changes on the cortical activity induced by 4-AP for the ictal and post-ictal periods are completely prevented by vinpocetine. Characteristic EEG recordings under control conditions (i.e. before the injection of 4-AP) in the animals pre-injected with vehicle and pre-injected with vinpocetine are shown on the top traces of Fig. 1a and b, respectively. In the animals pre-injected with vehicle, the changes induced by 4-AP on the EEG for the ictal (20 min) and post-ictal (30, 60 and 80 min) periods are shown in the recording below the control trace on Fig. 1a. In the animals preinjected with vinpocetine the changes on the EEG induced by 4-AP were practically cancelled (Fig. 1b). In order to have an objective measure of the cortical activity elicited by 4-AP during the post-ictal period, the EEG highest peak amplitude value was quantified in the animals that were pre-injected with vehicle and with vinpocetine. This EEG highest peak amplitude value is defined as the main positive to negative non-interrupted peak detected in 40 s of EEG recording (4 consecutive recordings of 10 s each). In the animals pre-injected with vehicle the EEG highest peak amplitude values after the injection of 4-AP significantly increase (Fig. 2a), whereas when the animals were pre-injected with vinpocetine the EEG highest peak amplitude values remain practically unchanged (Fig. 2b).
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Fig. 1. Vinpocetine inhibits the 4-AP-induced changes on the cortical activity for the ictal and post-ictal periods. (a) Representative EEG recordings taken in an animal pre-injected with vehicle before (top trace) and at the indicated times after the injection of 4-AP. (b) EEG recordings taken in the same animal 10 days after pre-injected with 2 mg/kg vinpocetine before (top trace) and at the indicated times after the injection of 4-AP.
3.2. Effect of vinpocetine on the abnormalities in amplitude and latency of the later ABR waves induced by 4-AP
3.3. Effect of vinpocetine on the change induced by 4-AP on P4 latency
In the animals pre-injected with vehicle, 4-AP progressively increased the amplitude of P3 (Fig. 3a) and progressively decreased the amplitude of P4 (Fig. 3c). These changes in P3 and P4 wave peak amplitudes induced by 4-AP were vinpocetine sensitive. In the animals preinjected with vinpocetine the progressive increase in P3 amplitude induced by 4-AP at specific times was abolished (Fig. 3b) and the decrease in P4 amplitude induced by 4-AP was markedly reduced (Fig. 3d). The amplitudes of the P1 and the P2 waves of the ABR were unchanged by 4-AP in the animals pre-injected with vehicle and in the animals pre-injected with vinpocetine. Fig. 4 shows representative ABR recordings elicited by the stimulus of 100 dB before and 1 h after the injection of 4-AP in an animal pre-injected with vehicle (Fig. 4a) and in the same animal pre-injected with vinpocetine 10 days after (Fig. 4b).
In the animals pre-injected with vehicle the latency of the P4 wave of the ABRs induced by 100 dB at the tone frequencies of 8 and 4 kHz is increased 1 h and 30 min after the injection of 4-AP until the end (100 min) of the experiment,
Fig. 2. Objective estimation of the cortical changes induced by 4-AP during the post-ictal period. Highest EEG peak amplitude values obtained before (0) and at the indicated times after the injection of 4-AP in the animals preinjected 1 h before 4-AP: (a) with vehicle or (b) with vinpocetine. Results are the meanGSEM values of 5 animals. *, P!0.05 between data obtained before and at the indicated time after 4-AP. a, P!0.05 between data obtained in the absence (vehicle) and in the presence of vinpocetine at equivalent times.
Fig. 3. Vinpocetine inhibits the 4-AP-induced abnormalities in the amplitude of the later ABR waves. P3 wave peak amplitude induced by a stimulus of 100 dB nHL at 8 kHz before (0) and 30, 60, 80 and 100 min after the injection of 4-AP in the animals pre-injected with vehicle (a) and in the animals pre-injected with vinpocetine (b). P4 wave peak amplitude induced by a stimulus of 100 dB nHL at 8 kHz before (0) and 30, 60, 80 and 100 min after the injection of 4-AP in the animals pre-injected with vehicle (c) and in the animals pre-injected with vinpocetine (d). Data are the meanGSEM values of 5 independent animals. *, P!0.05 between data obtained before and at the indicated time after 4-AP. a, P!0.05 between data obtained in the absence (vehicle) and in the presence of vinpocetine at equivalent times.
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Fig. 4. Representative ABR recordings taken before and 1 h after the injection of 4-AP in an animal pre-injected with vehicle (a) or with vinpocetine (b). The arrow indicates the onset time of the monaural pure tone stimulus (100 dB, 8 kHz). Each trace represents the average of 500 responses. The baseline is indicated by the dashed line.
respectively (left columns on Table 1). However, when the same animals were pre-injected with vinpocetine the increases in P4 latency induced by 4-AP at both tone frequencies were completely prevented (right columns on Table 1). The latencies of the other first three waves of the ABR, namely P1, P2 and P3, in response to the stimulus of 100 dB at both, 4 and 8 kHz, were unchanged by 4-AP (data not shown). 3.4. Vinpocetine inhibits the hearing loss induced by 4-AP The auditory thresholds for 8 and 4 kHz tone frequencies were tested before the injection of 4-AP and then 30 and 60 min after the injection of 4-AP in the animals pre-injected with vehicle and in the animals pre-injected with vinpocetine. Table 2 shows that the 4-AP-induced increase in the auditory threshold observed in the animals pre-injected Table 1 Effect of vinpocetine on the increases in P4 wave peak latencya induced by 4-AP Before
8 kHz Vehicle 3.48G0.06
8 kHz Vinpocetine 3.48G0.04
with vehicle at the two tone frequencies (left columns) is lost in the animals pre-injected with vinpocetine (right columns).
4. Discussion The present study shows that vinpocetine is an effective inhibitor of the epileptic cortical activity, as well as of the retro-cochlear alterations and the hearing loss induced by 4-AP in the guinea pig in vivo. The positive effect of vinpocetine on seizure control is indicated by the complete cancellation of all the epileptic manifestations induced by 4-AP, such as convulsions and EEG changes induced for the ictal and post-ictal periods. In the guinea pig, P3 and P4 are waves of the ABR generated in the medial and the lateral superior olivary nucleus, respectively (Wada and Starr, 1983a,b,c). Therefore the increase in P3 amplitude, the decrease in P4 amplitude and the increase in P4 latency observed here in the 4-AP animal model of epilepsy indicate alterations in Table 2 Vinpocetine inhibits the increase in the ABR thresholda induced by 4-AP at 8 and 4 kHz
4-AP (vehicle)
4-AP (vinpocetine)
30 min 60 min 80 min 100 min
3.51G0.13 3.80G0.10b 3.80G0.10b 3.83G0.12b
3.55G0.07 3.45G0.08 3.36G0.05c 3.35G0.07c
Before
Before
4 kHz Vehicle 3.50G0.02
4 kHz Vinpocetine 3.45G0.06
4-AP (vehicle)
4-AP (vinpocetine)
30 min 3.78G0.09b 3.37G0.06 3.54G0.11 60 min 3.78G0.09b 80 min 3.78G0.09b 3.56G0.04 100 min 3.75G0.10b 3.43G0.09 P4 latency in response to a stimulus of 100 dB at two tone frequencies (4 and 8 kHz). a Results are the averageGSEM values in ms of data obtained from 5 animals. Before refers to control values, i.e. before 4-AP injection. b P!0.05 between data before and at the indicated time after 4-AP in the same column at 8 kHz (upper columns) and at 4 kHz (lower columns). c P!0.05 between data in the same line (i.e. animals injected with vehicle and with vinpocetine).
8 kHz Vehicle 3.8G1.3
8 kHz Vinpocetine 2.5G1.4
4-AP(vehicle)
4-AP(vinpocetine)
30 min 60 min
10.0G2.0 23.8G6.3c
K1.3G2.4b 0.0G3.5b
Before
4 kHz Vehicle 13.8G1.3
4 kHz Vinpocetine 13.8G1.3
4-AP(vehicle)
4-AP(vinpocetine)
30 min 20G2.0 11.3G1.3b c 60 min 30G3.5 11.3G1.3b a Results are the averageGSEM values in dB nHL of data obtained from 5 animals. Before refers to control values at the specified frequency (4 or 8 kHz) before 4-AP injection. b P!0.05 between data in the same line (i.e. animals injected with vehicle and with vinpocetine). c P!0.05 between data before and at the indicated time after 4-AP in the same column.
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the physiology of the medial and lateral superior olivary nuclei. Taking into account that changes in the auditory nuclei of the superior olivary complex are reported to accompany (Fisman, 1975) or even precede (Kohsaka et al., 1999, 2001) the epileptic cortical activity in patients, it is likely that the inhibition of the 4-AP-induced alterations in those nuclei of the superior olivary complex by vinpocetine contributes to the its antiepileptic action. The failure of 4-AP in modifying the amplitudes and latencies of the earlier ABR waves (P1 and P2) indicates that the 4-AP-induced hearing decline (evidenced by the marked increase in the auditory threshold induced by 4-AP) does not involve damage of the more peripheral structures of the auditory pathway, such as the VIII nerve or the cochlear hair cells. While the finding that 4-AP markedly changes the amplitudes and latencies of the later ABR waves (P3 and P4) indicates that the 4-AP-induced hearing decline involves retro-cochlear damage. Our present findings showing that by eliminating the 4-APinduced alterations in P3 and P4 parameters with vinpocetine the hearing decline induced by 4-AP is also eliminated, supports the conclusion that the hearing loss observed in pharmacological animal models of generalised epilepsy is due to the alterations in the activity of the generators of the later ABR waves localised in the superior olivary complex (Nekrassov and Sitges, 2003). However, it is noteworthy that besides the protective action of vinpocetine against the hearing loss linked with disturbances in the physiology of nuclei of the superior olive generating the later waves of the ABR, vinpocetine also protects the aminoglycoside antibiotic-induced hearing loss, that results from alterations in the P1 wave of the ABRs (Nekrassov and Sitges, 2000). The hearing decline previously reported in two pharmacological animal models of epilepsy (Nekrassov and Sitges, 2003) and the tendency towards elevated ABR thresholds reported in patients suffering severe grand mal generalised epilepsy (Soliman et al., 1993) suggest that epilepsy per se causes hearing deficits, that may contribute to the deleterious cognition concomitant to epilepsy (Jokeit and Ebner, 1999; Meador, 2001; Prevey et al., 1998; Theodore et al., 1999), and the present results show that vinpocetine prevents both, the epileptic cortical activity and the accompanying hearing decline. Additionally, besides the hearing deterioration caused by epilepsy, several of the most commonly used antiepileptic drugs also cause alterations in the ABRs and hearing deficits (Armon et al., 1990; Chan et al., 1990; de la Cruz and Bance, 1999; Hirose et al., 1990; Mervaala et al., 1987; Yuksel et al., 1995; Zgorzalewicz and Galas-Zgorzalewicz, 2000), that may aggravate the cognitive deficits caused by the illness. Therefore the inhibition by vinpocetine of the ABR alterations and hearing decline that accompany the epileptic cortical activity shown here, suggest some advantages of vinpocetine for the treatment of epilepsy.
Acknowledgements The authors thank Tzipe Govezensky for her help in the statistical analysis of data.
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Vinpocetine prevents 4-aminopyridine-induced changes in the EEG, the auditory brainstem responses and hearing Maria Sitgesa,b,*, V. Nekrassova,b a
Depto. de Biologı´a Celular y Fisiologı´a, Instituto de Investigaciones Biome´dicas, UNAM, Apartado Postal 70228, Ciudad Universitaria 04510, Mexico City, DF, Mexico b Instituto Nacional de la Comunicacion Humana, CNR, SSA, Mexico, DF, Mexico Accepted 28 June 2004
Abstract Objective: The purpose of the present study was to investigate if the sodium channel blocker and memory enhancer, vinpocetine, was capable to overcome the epileptic cortical activity, the abnormalities in the later waves of the auditory brainstem responses (ABRs) and the hearing loss induced by 4-AP at a convulsing dose in the guinea pig in vivo. Methods: EEG and ABR recordings before and at specific times within 2 h after the injection of 4-AP (2 mg/kg, i.p.) were taken in animals pre-injected i.p. with vehicle or with vinpocetine (2 mg/kg) 1 h before 4-AP. The amplitude and latency of the ABR waves induced by a monoaural stimulus of high intensity (100 dB nHL) at 4 and 8 kHz pure tone frequencies and the ABR threshold were determined in the animals exposed to the different experimental conditions. Results: Vinpocetine inhibited the EEG changes induced by 4-AP for the ictal and post-ictal periods as well as the alterations in amplitude and latency of P3 and P4 and the increase in the ABR threshold induced by 4-AP. Conclusions: Vinpocetine prevents the retro-cochlear alterations and the hearing decline that accompany the epileptic cortical activity. Significance: Vinpocetine could be a promising alternative for the treatment of epilepsy. q 2004 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Antiepileptic drugs; Cognitive functions; Epilepsy; Lateral superior olive; Medial superior olive; Audition
1. Introduction Auditory brainstem responses (ABRs) are far fieldevoked potentials that consist of several waves that occur within 10 ms post-stimulus. In patients ABRs are useful in the clinical diagnosis of retro-cochlear lesions, because abnormalities of the later wave components of the ABRs are indicative of alterations in their generators that are localised in specific nuclei of the auditory brainstem (Hughes and Fino, 1985). Determination of the ABRs threshold is also an objective measure of the hearing sensitivity; because while * Corresponding author. Address: Depto. de Biologı´a Celular y Fisiologı´a, Instituto de Investigaciones Biome´dicas, UNAM, Apartado Postal 70228, Ciudad Universitaria 04510, Mexico City, DF, Mexico. Tel.: C525-5622-3866; fax: C525-5622-3897. E-mail address: [email protected] (M. Sitges).
the hearing sensitivity declines, stimuli of progressively higher intensity (in dB) are needed for evoking the ABRs. The epileptic cortical activity induced in the guinea pig by the convulsing agent 4-aminopyridine (4-AP) is accompanied by specific alterations of the later waves of the ABRs, that resulted in hearing decline (Nekrassov and Sitges, 2003). Glutamate is the most important excitatory neurotransmitter in the brain. Therefore, epilepsy can be induced by increasing the cerebral excitatory neurotransmission with drugs such as 4-AP, that in vivo has shown to increase particularly glutamate release (Morales-Villagran and Tapia, 1996). Many of the most widely used antiepileptic drugs suppress the abnormal neuronal excitability associated with seizures by means of complex voltage- and frequency-dependent inhibition of ionic currents through sodium channels (Lingamaneni and Hemmings, 2003;
1388-2457/$30.00 q 2004 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2004.06.019
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Ragsdale and Avoli, 1998). Vinpocetine (ethyl apovincamine-22-oate) is a sodium channel blocker (Erdo¨ et al., 1996), that selectively inhibits neurotransmitter release triggered by increased sodium channel permeability (Sitges and Nekrassov, 1999; Trejo et al., 2001). For instance, the release of glutamate induced by the activation of sodium channels with veratridine in cerebral isolated nerve endings is inhibited by vinpocetine (Sitges and Nekrassov, 1999), as well as by carbamazepine and other antiepileptic drugs (Ambrosio et al., 2000). In the guinea pig in vivo vinpocetine inhibits the alterations of some of the ABR waves and the hearing loss induced by the aminoglycoside antibiotic, amikacin (Nekrassov and Sitges, 2000). Also previous studies have shown that vinpocetine improves cognitive functions in rats (DeNoble, 1987; DeNoble et al., 1986), as in healthy human volunteers (Bhatti and Hindmarch, 1987; Subhan and Hindmarch, 1985). An elegant compilation of the basic and clinical pharmacology of the nootropic drug vinpocetine can be found in Bo¨no¨czk et al. (2000). Since one major problem of epilepsy is the deleterious cognitive consequences that, among other factors, are caused by the illness (Jokeit and Ebner, 1999; Meador, 2001; Prevey et al., 1998; Theodore et al., 1999), in the present study the antiepileptic potential of vinpocetine was explored by testing its effect on the epileptic cortical activity, the alterations on the later ABR waves and the hearing decline induced by the convulsing agent 4-AP in the guinea pig in vivo.
2. Methods Experiments were performed in pigmented adult male guinea pigs initially weighing 312G17 g. EEG recordings were used to evaluate changes in cortical excitability and ABR recordings to evaluate hearing. For EEG recordings, needle electrodes were placed subcutaneous over the left temporal area (active electrode) and over the left frontal area between the midline and the arched portion of the orbital crest (reference electrode). For ABR recordings, needle electrodes were placed subcutaneous at the ipsilateral left pinna (reference electrode), the contralateral pinna (ground electrode) and the vertex (active electrode). EEG and ABR recordings were performed in a sound proof room with a Nihon-Kohden Neuropack IV Mini (MEB-5304 K) system. Prior to each sequence of recordings, guinea pigs were anaesthetised with ketamine (50 mg/kg/10 mg/kg xylazine, i.p.) for restraining movement, stress and muscular activity. This anaesthetic was chosen because in the guinea pig it does not change the latency or the amplitude of the ABR waves or the ABR threshold within the time of the experiments (Nekrassov and Sitges, 2003). Recordings were taken following methods previously reported (Nekrassov and Sitges, 2000, 2003). For the ABR recordings monaural tone burst stimuli of 4 and 8 kHz were
delivered by TDH 39 earphone located 1 cm from the left ear. The right ear was blocked with a special wax plug that substantially reduced the sound level at this ear. Four or 8 kHz alternating polarity tone bursts (20/s), with 2 ms duration and 0.5 ms rise-fall times were used for evoking the potentials. Responses were amplified and averaged (500 responses), displayed vertex positive up and saved to disk for off-line analyses. The Institutional Animal Use and Care Committee approved all experimental procedures. 2.1. Animals and treatments 4-AP, obtained from Sigma (St Louis MO), was dissolved in oxygenated HEPES buffer (composition in mM: 127 NaCl, 1.18 KH2PO4, 3.73 KCl, 1 CaCl2, 1.18 MgSO4, 20 HEPES and 5.6 mM dextrose). The dose of 4-AP was chosen on the basis of preliminary experiments in non-anaesthetised animals injected with several doses (in mg/kg: 1, 2, 4 and 6) of 4-AP. In the guinea pig, the dose of 2 mg/kg 4-AP was chosen to carry out the experimental series of the present study because the tonic–clonic convulsions induced by this dose that appear about 20 min after the injection of 4-AP last for about 10 min and the animals recuperate few hours after the end of the experiments. In contrast, in the animals (seven) injected with 4 mg/kg 4-AP the duration of the convulsions lasted for more than 1 h, and all the animals (four) injected with 6 mg/kg 4-AP died in status epilepticus. In the animals injected with 1 mg/kg the tonic–clonic convulsions were rare. Vinpocetine (kind gift of Armstrong International) was dissolved in saline acidified with HCl and adjusted to pH 4 with NaOH. The dose of vinpocetine was chosen also on the basis of preliminary experiments. For instance, the effect of vinpocetine at increasing concentrations (range from 0.5 to 5 mg/kg) was first tested on the tonic–clonic convulsions induced by the injection of 2 mg/kg 4-AP in anaesthetised animals. Since from the dose of 2 mg/kg, the anticonvulsant effect of vinpocetine was clearly observed, this dose of vinpocetine was chosen to carry out the experiments in the anaesthetised animals. Vinpocetine was injected 1 h before 4-AP because when injected longer before (3–4 h) its inhibitory effect was less pronounced in some animals. In other preliminary experiments the effect of 5 mg/kg vinpocetine injected 1 h before a higher dose (4 mg/kg) of 4-AP was tested. Under this experimental conditions only in half of the animals vinpocetine clearly inhibited the severe epileptic cortical activity induced by the convulsing agent at this high dose. Five guinea pigs were used for the experimental series presented in this study. In a first set of experiments these animals were injected with vehicle 1 h before anaesthesia. Vehicle refers to the solution used to dissolve vinpocetine. For testing the effect of 2 mg/kg vinpocetine, a second set of experiments in which the same animals were injected with vinpocetine 1 h before anaesthesia was done 10 days after
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the first set of experiments. Ten minutes after the injection of the anaesthetic solution 3 types of recordings: (1) the ABR recordings elicited by a stimulus of high intensity (100 dB), (2) the ABR recordings for determination of the auditory threshold and (3) the EEG recordings, were obtained in the animals pre-injected with vehicle and, 10 days after, in the same animals pre-injected with vinpocetine. Immediately after getting those recordings, the animals were injected (i.p.) with 4-AP 2 mg/kg for obtaining a new series of recordings taken at specific times after 4-AP injection. For instance, the EEG recordings for the ictal period were taken about 20 min after the injection of 4-AP, and the ABR recordings evoked by 100 dB or the EEG recordings for the post-ictal period were taken 30, 60, 80 and 100 min after the injection of 4-AP. The recordings for determination of the ABR thresholds were taken 30 and 60 min after 4-AP. All the recordings were taken within the first 2 h after 4-AP injection taking into account the start and duration time of the ictal and post-ictal periods induced by the convulsing agent. 2.2. Determination of ABR wave parameters The latency and amplitude of each wave component of the ABR elicited by the stimulus of high intensity (100 dB) with pure tone frequencies of 4 or 8 kHz was measured in all the ABR recordings obtained under the different experimental conditions at the specified times. The latency of each ABR wave (in ms) refers to the time interval between the onset of the auditory stimulus and the positive peak of the wave. The onset of the stimulus is indicated by the vertical arrow at the bottom of the recordings on Fig. 4. The peak amplitude (in mV) of each wave of the ABR is the difference between the positive peak of the wave and the reference baseline (trace between the stimulus and the appearance of the first wave of the ABRs on Fig. 4). 2.3. Determination of the hearing sensitivity ABR recordings elicited by stimuli of progressively lower intensity (in dB) were taken for determining the hearing threshold in the animals exposed to the different experimental conditions. Threshold is defined as the lowest stimulus intensity (in dB) at which the P3 wave of the ABR could still be recorded in three consecutive trials (each trial equals the average response to 500 stimuli). For obtaining the auditory threshold a tone burst response induced with an intensity of 100 dB normal hearing level (nHL) was initially recorded. Then thresholds were determined for each stimulus by reducing the intensity at 20 and 10 dB nHL intervals, and then at 5 dB intervals down from a suprathreshold ABR recording to identify the lowest level at which reproducible waves could be recognised. The sound threshold level was also objectively estimated. The amplitude of the P3 wave at the peak was plotted against the corresponding sound intensity in order to construct
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input–output curves at 20, 10 and 5 dB nHL steps for 4 and 8 kHz. 2.4. Statistics The differences between results obtained before and at specific times after 4-AP and the differences between results obtained in the absence (vehicle) and presence of vinpocetine were considered statistically significant when P was less than 0.05 using a repeated measure analysis of variance and a post-hoc Tukey’s Studentised test. All data are expressed as meansGstandard error of the mean.
3. Results 3.1. Vinpocetine inhibits all the changes induced by 4-AP on the EEG All anaesthetised animals injected with 2 mg/kg 4-AP developed generalised seizures. The onset of seizure activity, characterised by repetitive high amplitude spikesharp wave activity in the EEG tracing appears about 20 min after the injection of 4-AP. This change on the cortical activity elicited by 4-AP during convulsions is followed by a pattern of cortical activity characterised by isolated spikes of higher amplitude. The time of duration of this later pattern of cortical activity (not accompanied by convulsions) is referred to as the post-ictal period. The changes on the cortical activity induced by 4-AP for the ictal and post-ictal periods are completely prevented by vinpocetine. Characteristic EEG recordings under control conditions (i.e. before the injection of 4-AP) in the animals pre-injected with vehicle and pre-injected with vinpocetine are shown on the top traces of Fig. 1a and b, respectively. In the animals pre-injected with vehicle, the changes induced by 4-AP on the EEG for the ictal (20 min) and post-ictal (30, 60 and 80 min) periods are shown in the recording below the control trace on Fig. 1a. In the animals preinjected with vinpocetine the changes on the EEG induced by 4-AP were practically cancelled (Fig. 1b). In order to have an objective measure of the cortical activity elicited by 4-AP during the post-ictal period, the EEG highest peak amplitude value was quantified in the animals that were pre-injected with vehicle and with vinpocetine. This EEG highest peak amplitude value is defined as the main positive to negative non-interrupted peak detected in 40 s of EEG recording (4 consecutive recordings of 10 s each). In the animals pre-injected with vehicle the EEG highest peak amplitude values after the injection of 4-AP significantly increase (Fig. 2a), whereas when the animals were pre-injected with vinpocetine the EEG highest peak amplitude values remain practically unchanged (Fig. 2b).
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Fig. 1. Vinpocetine inhibits the 4-AP-induced changes on the cortical activity for the ictal and post-ictal periods. (a) Representative EEG recordings taken in an animal pre-injected with vehicle before (top trace) and at the indicated times after the injection of 4-AP. (b) EEG recordings taken in the same animal 10 days after pre-injected with 2 mg/kg vinpocetine before (top trace) and at the indicated times after the injection of 4-AP.
3.2. Effect of vinpocetine on the abnormalities in amplitude and latency of the later ABR waves induced by 4-AP
3.3. Effect of vinpocetine on the change induced by 4-AP on P4 latency
In the animals pre-injected with vehicle, 4-AP progressively increased the amplitude of P3 (Fig. 3a) and progressively decreased the amplitude of P4 (Fig. 3c). These changes in P3 and P4 wave peak amplitudes induced by 4-AP were vinpocetine sensitive. In the animals preinjected with vinpocetine the progressive increase in P3 amplitude induced by 4-AP at specific times was abolished (Fig. 3b) and the decrease in P4 amplitude induced by 4-AP was markedly reduced (Fig. 3d). The amplitudes of the P1 and the P2 waves of the ABR were unchanged by 4-AP in the animals pre-injected with vehicle and in the animals pre-injected with vinpocetine. Fig. 4 shows representative ABR recordings elicited by the stimulus of 100 dB before and 1 h after the injection of 4-AP in an animal pre-injected with vehicle (Fig. 4a) and in the same animal pre-injected with vinpocetine 10 days after (Fig. 4b).
In the animals pre-injected with vehicle the latency of the P4 wave of the ABRs induced by 100 dB at the tone frequencies of 8 and 4 kHz is increased 1 h and 30 min after the injection of 4-AP until the end (100 min) of the experiment,
Fig. 2. Objective estimation of the cortical changes induced by 4-AP during the post-ictal period. Highest EEG peak amplitude values obtained before (0) and at the indicated times after the injection of 4-AP in the animals preinjected 1 h before 4-AP: (a) with vehicle or (b) with vinpocetine. Results are the meanGSEM values of 5 animals. *, P!0.05 between data obtained before and at the indicated time after 4-AP. a, P!0.05 between data obtained in the absence (vehicle) and in the presence of vinpocetine at equivalent times.
Fig. 3. Vinpocetine inhibits the 4-AP-induced abnormalities in the amplitude of the later ABR waves. P3 wave peak amplitude induced by a stimulus of 100 dB nHL at 8 kHz before (0) and 30, 60, 80 and 100 min after the injection of 4-AP in the animals pre-injected with vehicle (a) and in the animals pre-injected with vinpocetine (b). P4 wave peak amplitude induced by a stimulus of 100 dB nHL at 8 kHz before (0) and 30, 60, 80 and 100 min after the injection of 4-AP in the animals pre-injected with vehicle (c) and in the animals pre-injected with vinpocetine (d). Data are the meanGSEM values of 5 independent animals. *, P!0.05 between data obtained before and at the indicated time after 4-AP. a, P!0.05 between data obtained in the absence (vehicle) and in the presence of vinpocetine at equivalent times.
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Fig. 4. Representative ABR recordings taken before and 1 h after the injection of 4-AP in an animal pre-injected with vehicle (a) or with vinpocetine (b). The arrow indicates the onset time of the monaural pure tone stimulus (100 dB, 8 kHz). Each trace represents the average of 500 responses. The baseline is indicated by the dashed line.
respectively (left columns on Table 1). However, when the same animals were pre-injected with vinpocetine the increases in P4 latency induced by 4-AP at both tone frequencies were completely prevented (right columns on Table 1). The latencies of the other first three waves of the ABR, namely P1, P2 and P3, in response to the stimulus of 100 dB at both, 4 and 8 kHz, were unchanged by 4-AP (data not shown). 3.4. Vinpocetine inhibits the hearing loss induced by 4-AP The auditory thresholds for 8 and 4 kHz tone frequencies were tested before the injection of 4-AP and then 30 and 60 min after the injection of 4-AP in the animals pre-injected with vehicle and in the animals pre-injected with vinpocetine. Table 2 shows that the 4-AP-induced increase in the auditory threshold observed in the animals pre-injected Table 1 Effect of vinpocetine on the increases in P4 wave peak latencya induced by 4-AP Before
8 kHz Vehicle 3.48G0.06
8 kHz Vinpocetine 3.48G0.04
with vehicle at the two tone frequencies (left columns) is lost in the animals pre-injected with vinpocetine (right columns).
4. Discussion The present study shows that vinpocetine is an effective inhibitor of the epileptic cortical activity, as well as of the retro-cochlear alterations and the hearing loss induced by 4-AP in the guinea pig in vivo. The positive effect of vinpocetine on seizure control is indicated by the complete cancellation of all the epileptic manifestations induced by 4-AP, such as convulsions and EEG changes induced for the ictal and post-ictal periods. In the guinea pig, P3 and P4 are waves of the ABR generated in the medial and the lateral superior olivary nucleus, respectively (Wada and Starr, 1983a,b,c). Therefore the increase in P3 amplitude, the decrease in P4 amplitude and the increase in P4 latency observed here in the 4-AP animal model of epilepsy indicate alterations in Table 2 Vinpocetine inhibits the increase in the ABR thresholda induced by 4-AP at 8 and 4 kHz
4-AP (vehicle)
4-AP (vinpocetine)
30 min 60 min 80 min 100 min
3.51G0.13 3.80G0.10b 3.80G0.10b 3.83G0.12b
3.55G0.07 3.45G0.08 3.36G0.05c 3.35G0.07c
Before
Before
4 kHz Vehicle 3.50G0.02
4 kHz Vinpocetine 3.45G0.06
4-AP (vehicle)
4-AP (vinpocetine)
30 min 3.78G0.09b 3.37G0.06 3.54G0.11 60 min 3.78G0.09b 80 min 3.78G0.09b 3.56G0.04 100 min 3.75G0.10b 3.43G0.09 P4 latency in response to a stimulus of 100 dB at two tone frequencies (4 and 8 kHz). a Results are the averageGSEM values in ms of data obtained from 5 animals. Before refers to control values, i.e. before 4-AP injection. b P!0.05 between data before and at the indicated time after 4-AP in the same column at 8 kHz (upper columns) and at 4 kHz (lower columns). c P!0.05 between data in the same line (i.e. animals injected with vehicle and with vinpocetine).
8 kHz Vehicle 3.8G1.3
8 kHz Vinpocetine 2.5G1.4
4-AP(vehicle)
4-AP(vinpocetine)
30 min 60 min
10.0G2.0 23.8G6.3c
K1.3G2.4b 0.0G3.5b
Before
4 kHz Vehicle 13.8G1.3
4 kHz Vinpocetine 13.8G1.3
4-AP(vehicle)
4-AP(vinpocetine)
30 min 20G2.0 11.3G1.3b c 60 min 30G3.5 11.3G1.3b a Results are the averageGSEM values in dB nHL of data obtained from 5 animals. Before refers to control values at the specified frequency (4 or 8 kHz) before 4-AP injection. b P!0.05 between data in the same line (i.e. animals injected with vehicle and with vinpocetine). c P!0.05 between data before and at the indicated time after 4-AP in the same column.
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the physiology of the medial and lateral superior olivary nuclei. Taking into account that changes in the auditory nuclei of the superior olivary complex are reported to accompany (Fisman, 1975) or even precede (Kohsaka et al., 1999, 2001) the epileptic cortical activity in patients, it is likely that the inhibition of the 4-AP-induced alterations in those nuclei of the superior olivary complex by vinpocetine contributes to the its antiepileptic action. The failure of 4-AP in modifying the amplitudes and latencies of the earlier ABR waves (P1 and P2) indicates that the 4-AP-induced hearing decline (evidenced by the marked increase in the auditory threshold induced by 4-AP) does not involve damage of the more peripheral structures of the auditory pathway, such as the VIII nerve or the cochlear hair cells. While the finding that 4-AP markedly changes the amplitudes and latencies of the later ABR waves (P3 and P4) indicates that the 4-AP-induced hearing decline involves retro-cochlear damage. Our present findings showing that by eliminating the 4-APinduced alterations in P3 and P4 parameters with vinpocetine the hearing decline induced by 4-AP is also eliminated, supports the conclusion that the hearing loss observed in pharmacological animal models of generalised epilepsy is due to the alterations in the activity of the generators of the later ABR waves localised in the superior olivary complex (Nekrassov and Sitges, 2003). However, it is noteworthy that besides the protective action of vinpocetine against the hearing loss linked with disturbances in the physiology of nuclei of the superior olive generating the later waves of the ABR, vinpocetine also protects the aminoglycoside antibiotic-induced hearing loss, that results from alterations in the P1 wave of the ABRs (Nekrassov and Sitges, 2000). The hearing decline previously reported in two pharmacological animal models of epilepsy (Nekrassov and Sitges, 2003) and the tendency towards elevated ABR thresholds reported in patients suffering severe grand mal generalised epilepsy (Soliman et al., 1993) suggest that epilepsy per se causes hearing deficits, that may contribute to the deleterious cognition concomitant to epilepsy (Jokeit and Ebner, 1999; Meador, 2001; Prevey et al., 1998; Theodore et al., 1999), and the present results show that vinpocetine prevents both, the epileptic cortical activity and the accompanying hearing decline. Additionally, besides the hearing deterioration caused by epilepsy, several of the most commonly used antiepileptic drugs also cause alterations in the ABRs and hearing deficits (Armon et al., 1990; Chan et al., 1990; de la Cruz and Bance, 1999; Hirose et al., 1990; Mervaala et al., 1987; Yuksel et al., 1995; Zgorzalewicz and Galas-Zgorzalewicz, 2000), that may aggravate the cognitive deficits caused by the illness. Therefore the inhibition by vinpocetine of the ABR alterations and hearing decline that accompany the epileptic cortical activity shown here, suggest some advantages of vinpocetine for the treatment of epilepsy.
Acknowledgements The authors thank Tzipe Govezensky for her help in the statistical analysis of data.
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