Differential effects of benzodiazepines, including diazepam, clonazepam, Ro 5-. 4864 and devazepide, on lindane-induced toxicity. Proc West Pharmacol Soc ...
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2007, 58, 4, 641656 www.jpp.krakow.pl
D. MLADENOVIÆ , D. HRNÈIÆ , D. VUÈEVIÆ , T. RADOSAVLJEVIÆ , H. LONÈAR1
2
1
1
STEVANOVIÆ , J. PETROVIÆ , V. UIÆ , D. DJURIC , O. STANOJLOVIÆ 2
2
3
2
2
ETHANOL SUPPRESSED SEIZURES IN LINDANE-TREATED RATS. ELECTROENCEPHALOGRAPHIC AND BEHAVIORAL STUDIES
1 2
Department of Pathophysiology, School of Medicine, University of Belgrade;
Laboratory for Neurophysiology, Department of Physiology, School of Medicine,
University of Belgrade;
3
Serbian Academy of Sciences and Arts, Belgrade, Serbia
This study examines the effects of ethanol on lindane-induced seizures in rats. The
animals were divided into following groups: 1. saline, 2. DMSO (dimethylsulfoxide),
3. lindane dissolved in DMSO in the dose of 4, 6 or 8 mg/kg (L4, L6 and L8 groups,
respectively), 4. ethanol 2 g/kg administered 30 min prior to lindane (protected groups AL4, AL6 and AL8) and 5. ethanol alone (2 g/kg). In order to determine ethanol
concentration in plasma, blood samples were collected by cardiac puncture 30 and 60 min after ethanol injection. For EEG and power spectra recordings, electrodes
were implanted into the skull. The lindane treatment resulted in a dose-dependent increase of seizure incidence and severity. The rats displayed severe seizure patterns characterized by high voltage spike-wave complexes, poly-spikes and sleep-like patterns in EEG, while the power spectra were intensively elevated in comparison to
the corresponding controls. Ethanol alone led to increased EEG power spectra, which became dominant in the range of 0-4 Hz. For evaluation of anticonvulsant ethanol
action we compared latency to seizure, incidence and seizure severity (scale from 0 to 4) in the examined groups. Ethanol diminished seizure incidence in AL4 and AL6
groups, decreased intensity of convulsions, and prolonged duration of latency period in AL8 group. We observed suppression of the EEG signs of lindane-provoked
epileptiform activity in AL4 and AL6, but not in AL8 group. These results suggest that
ethanol acted protectively on lindane-induced seizures and suppressed behavioral and epileptic EEG spiking activity.
Keywords:
EEG, rats, lindane, ethanol, seizure
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INTRODUCTION
The effects of ethanol on epilepsy are very complex. While chronic ethanol abuse is occasionally followed by series of seizures during the withdrawal period, acute ethanol intake, exerts mainly inhibitory effects system
and
is
usually
associated
with
an
increase
on
of
the
central
seizure
nervous
threshold
(1).
Numerous in vivo studies showed that ethanol inhibits calcium influx through Nmethyl-D-aspartate
(NMDA)
glutamatergic
receptors
(2,
3)
and
that
ethanol
enhances the inhibitory action of GABA (4 - 6). However, in vitro studies of ethanol effects on GABA receptors led to equivocal results and while some authors observed enhanced GABA-gated currents (7), the results of the others did not support this finding (8). Ethanol administration resulted in elevation of the EEG bands in theta power. The extent of this elevation is dependent on blood alcohol level. This may be taken as an electrophysiological index of the balance between excitation and inhibition in the brain cortex (9). Stenberg et al. (10) also found that 1.0 g/kg ethanol increased EEG power in the theta and beta bands in humans. However, in experiments on adult rats, Ehlers et al. (11 - 13) reported that 0.75 g/kg ethanol, decreased the EEG power over all frequencies.
γ
Lindane ( -1,2,3,4,5,6-hexachlorocyclohexane), an organochloride pesticide is widely
employed
in
human
and
veterinary
medicine.
Lindane
represents
a
proconvulsant drug. When administered either orally or intraperitoneally, lindane evoked tonic and clonic seizures in a dose-dependent manner (14, 15). Lindaneinduced seizures are frequently used as an experimental model for testing protective effects of antiepileptic drugs (16, 17). Lindane also decreases the seizure threshold in electrically kindled animals (18). These lindane effects are mediated by the blockade of GABA receptors in the brain (19). Therefore, central effects of both ethanol and lindane are related to similar mechanisms in opposite direction. The data on ethanol effects on lindane-induced epileptic activity under EEG monitoring are still not sufficiently reported in the currently available literature. This prompted us to study the influence of ethanol and lindane distributed alone, as well as in combination on bioelectric brain activity and behavior of adult rat males.
MATERIALS AND METHODS
Animals We
used
Academy
2-month-old
Breeding
Wistar
Laboratories,
rat
adult
Belgrade.
males They
(170 were
-
200
kept
g),
raised
under
at
Military
controlled
Medical
environmental
conditions (22 ± 1 °C temperature, 50% relative humidity and 12/12 h light/dark cycle with light switched on at 9 a.m), and were housed individually with free access to standard laboratory animal chow and tap water. A total of 91 animals were divided into five main groups: 1. control, saline-injected (n = 6); 2. DMSO-treated (n = 6); 3. lindane-administered L4 (4 mg/kg; n = 11), L6 (6 mg/kg; n = 12) and L8 (8 mg/kg; n = 12); 4. ethanol-injected (2.0 g/kg) 30 min prior to lindane, referred to as to the
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ethanol-treated groups AL4, (n = 7); AL6, (n = 13) and AL8 (n = 18), which were serving to evaluate the ability of ethanol to inhibit lindane-induced seizures and 5. ethanol alone (2.0 g/kg, n = 6). Each rat was used only once. For i.p. administration lindane was dissolved in dimethylsulfoxide (DMSO) and injected in a volume of 0.5 ml/kg body weight.
Surgery and EEG recordings For EEG recordings, rats were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and three gold-plated electrodes were implanted over frontal, parietal and occipital cortices by stereotaxic method. The animals were left to recover 7 days after the surgery. EEG apparatus (Alvar, France) with a modified output degree enabling to transfer output signals to the input circuit of an 8-channel, 12-byte AD card PCL-711B (Advantech, Co. Ltd, Taiwan) installed into a computer and the corresponding software were used (20, 21). Frequency range was defined by the time constant (0.3 seconds, lower and upper limit frequencies of 0.5 and 30 Hz, respectively). The power spectra were plotted and the integrated energy signals were expressed as µV . Upon completion of the 60-minute recording sessions, 2
the animal would be removed from the recording chamber and returned to its home cage. All
experimental
procedures
were
carried
out
in
accordance
with
The
European
Council
Directive (86/609/EEC) and approved by The Ethical Committee of the University of Belgrade.
Assessment of seizure activity The animals put into separate transparent plastic cages (55x35x15 cm) were observed during 30 min test-time for the occurrence of seizure behavior signs. Seizure behavior was assessed by incidence of motor seizures and seizure severity grade determined after Fischer and Kittner (22) and modified using a descriptive rating scale from 0 - 4 (0 - no response; 1 - head nodding, lower jaw twitching; 2 - myoclonic body jerks, bilateral forelimb clonus with full rearing; 3 - progression to generalized clonic convulsions followed by tonic extension of fore- and hind limbs and tail; 4 status epilepticus). The rats were considered protected from lindane-induced seizures, if they did not develop clonus or tonus within 30 min of test-time which included the observing period after administration. Mortality was recorded after lindane administration. In addition, latency to seizure, defined as a time from lindane injection to the first seizure response, was also recorded.
Plasma level of ethanol Blood
samples
were
obtained
by
cardiac
puncture
under
light
ether
anesthesia.
Plasma
concentrations of ethanol were determined 30 and 60 min after i.p. ethanol injection. Controls received physiological saline by the same route. Heparinized blood samples were centrifuged (3.000 rpm, 5 min) and 0.1 ml aliquots of the blood plasma transferred into dry test tubes and ethanol concentration determined using RANDOX kit (RANDOX Laboratories Ltd., Ardmore, UK) as suggested by the supplier. Ethanol concentrations were expressed in mmol/l as means ± SEM (12 - 14 rats for each group).
Data analysis Significance of the differences in the incidence of seizures was evaluated using Fishers exact probability test. Since the normal distribution of the data on seizure severity grade and latency has not
been
estimated
by
Kolmogorov-Smirnov
test,
therefore
the
non-parametric
analyses
were
employed. Kruskal-Wallis ANOVA and Mann Whitney U-test were used to determine the statistical significance of the differences between the groups in median seizure grade and median duration of
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the latency (*p
