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. 2007 Oct 24;27(7):923–932. doi: 10.1007/s10571-007-9222-5

Interaction of Delta Sleep-inducing Peptide and Valproate on Metaphit Audiogenic Seizure Model in Rats

Olivera Stanojlović 1,✉, Dragan Hrnčić 1, Aleksandra Rašić 1, Helena Lončar-Stevanović 1, Dragan Djuric 1, Veselinka Šušić 2
PMCID: PMC11517280  PMID: 17957464

Abstract

Effects of valproate (VPA), a conventional antiepileptic drug and natural delta sleep-inducing peptide (DSIP) on metaphit (1-[1-(3-isothiocyanatophenyl)-cyclohexyl]-piperidine)-induced audiogenic reflex epilepsy were studied. For the purpose of the study, valproate in the doses of 50 or 75 mg/kg and DSIP (1.0 mg/kg) was i.p. injected either alone or in combination to adult Wistar male rats with fully developed metaphit seizures after eight audiogenic testing. The animals were stimulated using an electric bell (100 ± 3 dB and 5–8 kHz, for 60 s) 60 min after metaphit injection and afterwards at hourly intervals during the experiment. For EEG recording and power spectra analysis, three gold-plated screws were implanted into the scull. In EEGs of metaphit-treated animals polyspikes, spike-wave complexes and sleep-like patterns were recorded, while the power spectra were increased. Combined treatment of metaphit-induced seizures with valproate and DSIP was more effective than drugs alone especially during 4 h after administration. None of the applied dose combinations eliminated the EEG signs of metaphit-provoked epileptiform activity. Taken together, the results of the present study suggest that the combinations of valproate and DSIP should be considered as beneficial polytherapy in metaphit model of epilepsy.

Keywords: Audiogenic seizures, DSIP, Metaphit, Rat, Valproate

Introduction

Since monotherapy, a gold standard in epilepsy treatment for the last 20 years, failed, introduction of combination therapy attempted to improve effectiveness (Deckers et al. 2001). Animal models of epilepsy are of considerable interest for investigation of susceptibility to antiepileptic drugs (AED) and exploration of different drug combinations and their way of action in order to get better antiepileptic results with minimum adverse effects.

It is known that alternations in inhibitory and excitatory neurotransmission play a central role in the etiology of epilepsy and the over stimulation of glutamate receptors produce this disease. Metaphit (1-[1-(3-isothiocyanatophenyl)-cyclohexyl]-piperidine), a phencyclidine analog, a compound known to increase susceptibility to audiogenic seizures, has been investigated for more than two decades (Rafferty et al. 1985; Debler et al. 1989; Šušić et al. 1991). Metaphit opens the ion channel under the control of the NMDA receptor complex and enhances glutamate affinity for this receptor (Lipovac et al. 1993, 2003).

Various neurotransmitter and neuromodulatory systems found in CNS like endogenous anticonvulsants contribute to seizure threshold elevation and epileptogenesis delay (Bahh et al. 2005). Anti-excitatory and anti-epileptic role is general principle of neuropeptides as endogenous modulators of seizure activity (somatostatin, neuropeptide Y, galanin) (Baraban 2004). The same holds true for neuropeptide delta sleep-inducing peptide (DSIP). This nonapeptide, member of the natural sleep peptide family, has attracted attention in recent years as an endogenous modulator of epileptic activity. Due to its antiepileptic action in several acute generalized epilepsy forms (Prudchenko et al. 1993; Shandra et al. 1993, 1998), this natural peptide arose as a special interest of our research.

Valproate (2-propylpentanoic acid, VPA) was firstly recognized 40 years ago. It demonstrated efficacy in treatment of partial and generalized seizures, in animals and humans, and because of that, it is classified as one of the first-line antiepileptic drugs. Up to the present, great attention was given to the exploration of the ideal combination of two or more antiepileptic drugs when monotherapy is not effective. That is the reason for combining VPA with a lot of conventional and other antiepileptic drugs in various experimental models of epilepsy (Luszezki et al. 2006; Cuadrado and Armijo 2005).

In order to test drug combination providing long-term effect of VPA and DSIP in developing metaphit seizure we investigated the time course and characteristics of drugs that may act beneficially.

Methods

Adult male Wistar rats (170–200 g), bred at the Military Medical Academy Breeding Laboratories, Belgrade (Serbia), were used for this study. The animals were maintained under controlled environmental conditions (22–24°C, 50–60% humidity, 12 h/12 h light/dark cycle, light on at 9 a.m.) with free access to standard laboratory chow and tap water. They were housed individually in transparent plastic cages (55 × 35 × 15 cm).

Audiogenic stimulation (AGS) was applied for 60 s using an electric bell positioned on the top of the cage (100 ± 3 dB, 5–8 kHz frequency). None of the untreated animals screened for audiogenic susceptibility expressed seizure activity. The first stimulation was applied 60 min after metaphit administration and repeated thereafter at hourly intervals during the experiment for about 16 h. Audiogenic convulsive behavior was assessed by incidence of motor seizures and seizure severity grade determined as previously reported (Šušić and Marković 1993), using a descriptive rating scale ranging from 0–3 (0—no response; 1—wild running only; 2—wild running followed by clonic seizures; 3—wild running progressing into generalized clonic convulsions followed by tonic extension of fore- and hind limbs and tail). Incidence of seizures was defined as number of convulsing animals versus total number of animals in each group (as percentage). All experimental procedures were carried out in accordance with The European Council Directive (86/609/EEC) and national law and also approved by The Ethical Committee of the University of Belgrade.

The rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), positioned in a stereotaxic apparatus, and three gold-plated recording electrodes were implanted over frontal, parietal, and occipital cortices. The animals were adapted to the recording cable for three days. The cable allowed free movement during the normal behavior and epilepsy state for purpose of EEG registration. An EEG apparatus (RIZ, Zagreb, Croatia) with a modified output degree enabling the transfer output signals to the input circuit of 8-channel, 12-byte AD card PCL-711B (Advantech Co. Ltd.) installed into a computer, and the corresponding software were used.

Epochs for EEG analyses depended on characteristic EEG changes and lasted for about 10 s. EEG recordings for 24 h per day have been performed continuously.

The animals were divided into following experimental groups: (1) Saline-injected (n = 6); (2) Metaphit-administered (mp, 10 mg/kg, n = 14,); (3) Blocking VPA groups pretreated with mp (10 mg/kg), and after 8 h, administered VPA in the doses of 50 mg/kg (VPA50 group, n = 8) and 75 mg/kg (VPA75 group, n = 8); (4) Blocking DSIP group treated with mp (10 mg/kg), and after 8 h administered DSIP in the dose of 1 mg/kg (DSIP group, n = 14); (5) Combined groups treated with mp (10 mg/kg), and after 8 h, administered VPA 50 mg/kg + DSIP 1.0 mg/kg, (n = 8) or VPA 75 mg/kg + DSIP 1.0 mg/kg, (n = 8).

In order to investigate the effects of VPA and DSIP on fully developed seizure, only metaphit-treated animals displaying seizure in eight previous tests (Stanojlović et al. 2000a, 2006) received VPA, DSIP or their combination. The rats were exposed to AGS at hourly intervals. The injecting solutions given i.p. in a total volume of 0.1 ml were prepared in sterile physiological saline. VPA dose of 50 and 75 mg/kg was chosen as subeffective dose in seizures induced by penthylentetrazole and amygdala kindled (Cuadrado and Armijo 2005; Borowicz and Czuczwar 2003).

Chimney Test

In order to investigate the effects of VPA (50 mg/kg, n = 6; 75 mg/kg, n = 6), DSIP (1.0 mg/kg, n = 6), or their combination on rat motor coordination, the chimney test of Boissier et al. (1960) was used 1 h after injection. Briefly, animals had to climb backward up a plastic, transparent tube (inner diameter 7 cm, length 25 cm), and motor impairment was characterized by the percentage of animals unable to climb backward up the tube within 60 s.

Statistical Analysis

Significance of the differences was assessed between the groups. To evaluate the differences in the incidence of convulsive components, Fisher’s exact probability test was used. The seizure stage was statistically compared by the Kruskal-Wallis one-way ANOVA and Mann Whitney U-test. The quantification of total voltage power and of single frequency bands was done by employing a Berg-Fourier analyzer. The power spectra were plotted and the integrated energy signals expressed were μV2/Hz.

Substances

Metaphit methanesulfonate and DSIP are products of Sigma-Aldrich Chemical Co., USA, Valproate sodium was a kind gift of Hemofarm Pharmaceutical Works (Vršac, Serbia).

Results

In control saline-injected animals, AGS provoked neither behavioral (animals expressed normal gross behavioral activity) convulsive response nor changes in EEG recordings. Baseline state was characterized by desynchronization and high frequency low amplitude waves (9–12 Hz), while total power spectra showed the intensity of 350 μV2 (Fig. 1a). All metaphit-treated (10 mg/kg) animals express abnormalities in EEG, few hours upon administration in form of isolated spikes in the beginning and thereafter, broad sharp waves and occasional multispike complexes were noted (Fig. 1b). Synchronized, high-amplitude and low-frequency spikes (6–8 Hz), and sleep-like patterns were present in EEG of metaphit-treated rats during AGS with behaviorally noted complete motor seizure response of grade 3 (Fig. 1c). Spectrum power analysis of the electrocortical changes induced by metaphit revealed an increased total voltage power (8,000–10,000 μV2). VPA (75 mg/kg) + DSIP (1 mg/kg) led to a rapid behavioral but not electricortical reduction in metaphit-treated animals (Fig. 1d). EEG epileptiform activity was not abolished either by VPA or DSIP, or by their combination.

Fig. 1.

Fig. 1

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EEG records (left tracings) from the left frontal—right parietal cortical leads. Right-sequential power spectra of the corresponding EEG activity on the left (μV2): (a) control rats (baseline state without signs of epileptiform activity), (b) isolated spiking activity observed during few hours upon metaphit administration, (c) EEG record of maximal motor seizure response (grade 3) during sound stimulation (100 ± 3 dB, 60 s) in a metaphit-treated rat, irregular low-frequency synchronized spikes (6–8 Hz), and the corresponding intensive power spectra exceeding about three times of the baseline, (d) EEG changes on AGS in metaphit-treated rats 1 h after injection of the VPA (75 mg/kg ) and DSIP (1.0 mg/kg, i.p.) drug combination. Note isolated spiking activity and mildly reduced power spectra Time calibration 1 s; amplitude calibration 50 μV

Only those metaphit animals expressing epilepsy with a maximum incidence and seizure severity after the eighth AGS received VPA, DSIP, or their combination. VPA in the subeffective dose of 50 mg/kg was not able to change both behavioral and EEG seizure manifestation induced by metaphit. However, VPA in combination with 1 mg/kg DSIP (VPA50 + DSIP), reduced the number of seizure animals in all check points, especially 13 h after metaphit when half of the animals on AGS did not respond. Significant reduction in number of seizure animals was observed between VPA50 + DSIP and VPA50 group (e) in 9, 10, 12 (P < 0.01), and 13 h (P < 0.05) after metaphit injection. The reduction in the number of seizure animals by VPA50 + DSIP as compared to DSIP alone (h) group was significantly greater in 9 h (P < 0.05). Also VPA50 + DSIP reduced seizure intensity in 9 h (P < 0.01) and 11 h (P < 0.05) after metaphit injection more effectively than DSIP group (Figs. 2 and 3).

Fig. 2.

Fig. 2

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The reduction of incidence of metaphit seizures expressed in percentage after 8 h followed by different valproate doses (50 and 75 mg/kg) either alone or with DSIP (1 mg/kg) in drug combination. All animals were exposed to an intense AGS (100 ± 3 dB, 60 s) at hourly intervals after the injection. Y-axis—reduction of seizure incidence s; X-axis—time (h) after metaphit administration. For the comparison of the number of rats with convulsing activity between groups, we used Fisher`s exact probability test (*P < 0.05, **P < 0.01): mp—metaphit (10 mg/kg); VPA50−mp + valproate 50 mg/kg; VPA75−mp + valproate 75 mg/kg; DSIP−mp + DSIP, and two combination groups VPA50 + DSIP−mp + valproate 50 mg/kg + DSIP and VPA75 + DSIP−mp + valproate 75 mg/kg, +DSIP. Comparison of the number of rats convulsing between (a–j): a—mp vs. VPA75; b—mp vs. VPA75 + DSIP; c—VPA50 vs. VPA75; d—VPA50 vs. DSIP; e—VPA50 vs. VPA50 + DSIP; f—VPA50 vs. VPA75 + DSIP; g—VPA75 vs. DSIP; h—DSIP vs. VPA50 + DSIP; i—DSIP vs. VPA75 + DSIP; and j—VPA50 + DSIP vs. VPA75 + DSIP

Fig. 3.

Fig. 3

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Time course of mean seizure grade reduction upon metaphit (10 mg/kg i.p.) injection followed by VPA, DSIP, and their combination after 8 h. All animals were exposed to an intense AGS (100 ± 3 dB, 60 s) at hourly intervals after the injection. Severity of seizures (expressed as a seizure score) was determined by a descriptive rating scale ranging from 0 to 3 (0—no response; 1—wild running only; 2—clonic seizures; and 3—tonic extension). Comparison of mean seizure grades between experimental groups was evaluated by Kruskal Wallis ANOVA test (*P < 0.05, **P < 0.01). X axis—time after metaphit administration (h) Y axis—reduction of seizure intensity in percentages; For more details see Fig. 2

In VPA75 group the maximal reduction in number of convulsing animals and seizure intensity was noted 12 h after metaphit (up to 70%). The number of convulsing animals significantly decreased in VPA75 group compared to mp group (a) 10 h (P < 0.05) and 12 h (P < 0.05); and compared to DSIP (g) group 9 (P < 0.01), 10, and 11 h (P < 0.05) after metaphit injections. VPA75 significantly reduced seizure severity compared to: mp (a) in 9, 11, and 12 h (P < 0.05); VPA50 (c) at 11 h (P < 0.05), and DSIP (g) group 9–11 h (P < 0.05) after metaphit administration (Figs. 2 and 3).

In fully developed convulsions, DSIP in combination with VPA doses (50 and 75) decreased seizure incidence and running + clonic + tonic seizures intensity in comparison with the metaphit (b), VPA (f), and DSIP group (h, i), as shown in Figs. 2 and 3. The combination of VPA (75 mg/kg) and DSIP (1 mg/kg) led to profound protective activity with regard to all seizure parameters. Antiepileptic combination VPA75 + DSIP made numerous significant differences especially for the period of 4 h after administration. A complete absence of seizure incidence was observed during the 9th and 12th hour of AGS tests (Fig. 2). This combination was sufficient to suppress the number of rats with seizures in 9, 10, and 12 h AGS test in comparison with mp (b, P < 0.01), DSIP (i, P < 0.01), and VPA50 + DSIP (j, P < 0.05) groups and in comparison with VPA50 group (f) in 13 h after metaphit (P < 0.05) (Fig. 2).

The same holds true for VPA75 + DSIP combination group concerning reduction of convulsive intensity of metaphit-induced epilepsy (Fig. 3). The effect was statistically significant in comparison with: mp (b, P < 0.01), DSIP only group (i, P < 0.01), VPA50 + DSIP (j, P < 0.05) group in same AGS testings: 9, 10, and 12 h.

Chimney Test

There were no statistically significant differences in ability to perform the test between saline-injected rats and those treated with any of the applied drugs or their combinations (Table 1).

Table 1.

Effects of valproate, DSIP, and their combination on the motor coordination of rats in the chimney test

Treatment (mg/kg, i.p.) N Animals impaired (%)
Saline 6 0
Valproate (50) 8 0
Valproate (75) 8 12.5
DSIP (1) 8 0
Valproate (50) + DSIP (1) 8 0
Valproate (75) + DSIP (1) 8 12.5
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The data represent percentage of rats that failed to perform the chimney test within the 60 s observation period. Statistical analysis of the data was done by means of Fisher’s exact probability test. N: number of rats

Discussion

Here we showed that neither DSIP (1.0 mg/kg) nor VPA (50 mg/kg) administered alone expressed statistically significant effect on the seizure manifestations in metaphit-treated rats. However, combination of these drugs significantly diminished the number of convulsing animals and reduced seizure components (running, clonus, and tonus) especially during 4-h-period after administration. This finding shows that DSIP, an endogenous natural, somnogenic peptide enhances the anticonvulsive properties of non-protective VPA doses in metaphit-induced seizures (Figs. 2, 3). Antiepileptic potency of this drug combination expressed as abolition of tonic seizure component (mean seizure grade) can be compared with the effect of a higher VPA dose given alone (100 mg/kg) (Stanojlović et al. 2006; Hrnčić et al. 2006).

Administrations of VPA, DSIP, or any of their combinations did not impair motor coordination of rats as judged by the results of the chimney test (Table 1). This is in accordance with results concerning VPA acquired by Borowicz and Czuczwar (2003) and with Schoenenberger and Schneider-Helmert (1983) who showed that even a maximal dose of DSIP (50 mg/kg) produced no adverse side effect. These findings indicate that DSIP, as a natural peptide, did not potentiate neurotoxicity of VPA, a conventional antiepileptic drug, and therefore, underline the potential benefits of combining these drugs in future clinical studies and practice.

The evaluation of EEG abnormalities (large amplitude spikes, sharp waves, and intense power spectrum) obtained following an injection of metaphit 10 mg/kg and 8 h thereafter of VPA alone in effective antiepileptic dose of 100 mg/kg (Stanojlović et al. 2006), expressed similar epileptiform activity with our EEG records in animals from VPA75 + DSIP group. This finding is opposite with data reported by Nistico et al. (1980) who showed that 100 mg/kg of VPA reduced and eliminated cefazolin induced spikes. The action against spread of epileptic activity into motor system is demonstrated by the appearance of spiking activity in EEG without any motor behavioral seizure correlate after drugs combination, prominently in the period of AGS. On the other hand, we previously observed (Stanojolović et al. 2000b) that the peak in EEG delta power was registered during 2–11 h following DSIP administration (1 mg/kg, i.p.).

As far as in 1945, Allen (1945) described reflex or stimulus-sensitive epilepsy and metaphit we used along with intense audiogenic stimulus represents reflex epilepsy model suitable for the research of anticonvulsive agents (Debler et al. 1989; Chen et al. 1994).

Our current experiment is in agreement with previous work (Stanojlović et al. 2000a, 2006) and demonstrates that metaphit elicited audiogenic seizures whose incidence and severity increased with time reaching a peak about 8 h after administration. Time course of metaphit-induced seizure, which is consequence of long-lasting alterations in voltage-dependent Na+ channels, showed a peak at 18–24 h after injection in doses of 20 mg/kg after either i.v. or retro orbital injection (Debler et al. 1989), or 50 mg/kg in guinea pig (Lipovac et al. 1993), rats, and mice (Šušić et al. 1991).

Our results are in agreement with Chen et al. (1994), who demonstrated that the metaphit action in a single dose (10 and 20 mg/kg) after 8 h produced a maximal seizure activity, reduced the threshold current, and shortened the latency for clonus. The animal injected with metaphit would be “primed,” and when threshold is reached by a strong AGS in the animals would be convulsive, and it would facilitate synaptic potentials and accelerate limbic kindling enhancing excitability in forebrain, brainstem, and hippocampus (Chen et al. 1994).

The maximum release of transmitters occurred during intense AGS and after that, tissue stores of classical transmitters rapidly reconstituted, and then ribosomal synthesis filled out neuropeptide stores (Albanese et al. 1995). Various neurotransmitters and neuromodulatory systems have been found to enhance inhibitory brain system, and act as endogenous anticonvulsants. DSIP, an endogenous anticonvulsive substance, is constantly present in different brain regions, especially in the limbic structure, and can inhibit a number of the effects of excitatory amino acids (Umriukhin et al. 2004), preventing occurrences of the excitatory effect of the glutamate via NMDA-receptors and blocks calcium influx, in other words stopped excitotoxicity (Umriuhkin 2002; Sudakov et al. 2004). Russian neuroscientists described DSIP antiepileptic activity on bicuculine, corasol, picrotoxin, and benzylpenicillin sodium (all are associated with gamma-aminobutyric acid) induced epilepsy (Prudchenko et al. 1993; Shandra et al. 1993, 1998; Mikhaleva et al. 1992; Mendzheritskii et al. 1997). Taken together with the facts that DSIP after i.p. administration penetrates the blood–brain barrier and has no harmful effects when maximal dose is applied, obtained data put it in line of promising antiepileptics (Stanojlović et al. 2005). VPA has a number of not only minor (nausea, vomiting, heartburns), but also some serious side effects such as hepatotoxicity and teratogenicity (Ahmad et al. 2005). VPA and DSIP involve multiple ways of action including alterations on neurotransmitters and increase GABA synthesis by activating glutamic acid decarboxylase (GAD), which converts glutamic acid to GABA in rodents, (Mikhaleva et al. 1992; Mendzheritskii et al. 1997; Donnell et al. 2003) thus increasing inhibitory and decreasing excitatory amino acid levels in brain cortex. Shandra et al. (1998) demonstrated the existence of a link between DSIP and NMDA excitatory receptor. This supports the view that DSIP plays a neuroprotective role against NMDA and glutamate as an excitatory amino acid, which shares the same receptor with metaphit, although they bind at different binding sites. On the other hand, VPA suppresses NMDA evoked depolarization and can inhibit voltage-gated sodium channel (Steppuhn and Turski 1993).

In conclusion, the results of the present study suggest that combinations of valproate and DSIP should be considered beneficial polytherapy in the metaphit model of epilepsy. This experimental data may be helpful for predicting which drug combinations may prove effective in epileptic patients.

Acknowledgment

This work was supported by the Ministry for Science, Technology and Environment Protection of Serbia (Grant N#145029B).

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