ARTICLE
Auteur(s) : Nicole Pages1,2,
Pierre Maurois1, Geneviève Agnani3, Joseph
Vamecq4, Évelyne Fénart5, Pierre
Bac1, Bernadette Delplanque3
1Neuropharmacology Laboratory, Faculty of Pharmacy,
Châtenay Malabry
2Toxicology Laboratory, Faculty of Pharmacy,
Illkirch
3NMPA, Univ ParisXI, Orsay
4INSERM UNIV 045131, Neuropaediatrics, Salengro
Hospital, CHRU, Lille
5ONIDOL, Paris, France
Introduction
In the last decade, different in vivo and in vitro studies have
demonstrated the beneficial effect of polyunsaturated fatty acids
(PUFA) and specifically omega3 on cardiac and neuronal
excitability. This protective effect may be of clinical relevance
in the prevention of both cardiovascular and brain dysfunctions
including epileptic seizures [1, 2].
Among omega3, alphalinolenic acid (18:3n-3, ALA) was shown to be
protective against both arrythmia and ischemia [3-6]. ALA is found
in vegetable oils like rapeseed, soya, nuts, or linseed and
represents 9% of the highly monounsaturated (60%) rapeseed oil
whereas it is absent in polyunsaturated/omega6 rich sunflower or
corn oils.
The aim of the present paper was to study whether dietary
rapeseed oil could be of interest against audiogenic seizures in
magnesium-deficient mice (MDDAS test: Magnesium
Deficiency-Dependent Audiogenic Seizure test). This test is a
pluripotent model of epilepsy also allowing the detection of
neurotoxic or neuroprotective activities. The MDDAS test has been
validated previously [7] in adult magnesium-deficient mice
individually exposed to a calibrated audiogenic stimulus
(100 dBA, 10 kHz, 15 sec). It is characterized by 4
successive steps: Latency, Wild Running, Convulsions (Tonic
Seizure) and Recovery, the duration of which is recorded as a
mechanistic approach. Neurotoxic treatments decrease the
1st and 2nd phases and increase the
3rd and 4th phases. Neuroprotective
treatments using various anti epileptic drugs showed different
patterns according to their main mechanism of action, either
phenitoinergic or GABAergic, antioxidative or anti-inflammatory
[7].
Materials and methods
The investigation conforms to the Guide for the Care and Use of
Laboratory Animals published by the US National Institute of Health
(NIH, No 85-23, revised 1996). Female Swiss OF1 mice, were
purchased from Janvier (Le Genest-St-Isle, France) and divided into
two groups (n = 14). Each group was fed for 30 days, Mg-deficient
diets (50 ± 5 mg/kg) prepared as previously described [8],
containing 5% vegetable oils, either rich in ALA (rapeseed) or poor
in ALA (corn/sunflower 3:1). They were placed five per cage and
maintained on a 12h light-dark cycle at 21 ± 1° C. They had
free access to food and, in order to avoid an additional input,
distilled water.
At the end of the deprivation period, the body weight gain was
measured. The mice were afterwards transferred individually in a
plexiglass cage in an Apelex type 01-1668B actimeter (Bagneux,
France) and allowed to explore for a 3 minute period. Their
locomotor activity was measured by the crossing of the photocell
activity meter and automatically recorded. The experiment was
carried out in a sound proof room between 9: 00 and 13: 00 to
reduce the confounding influence of diurnal variation in
motility.
Maximun ElecroShock test (MES) was induced via a pair of
auricular clip electrodes by means of an electroshock stimulator
(Karl Kolbe, Scientific Technical Supplies, Frankfurt, Germany).
MES test measured the capacity of a test compound to provide
complete protection against threshold seizures (tonic hindlimb
extension in 100% of mice followed by clonic seizures) induced by
4 mA, 0.2 sec duration, 50 Hz, sinewave form. It allows
the detection of drugs acting on Na+ voltage-dependent
channels. For the MDDAS test, individual animals were placed in a 9
dm3-volume test chamber (30, 20 and 15 cm for
length, width and height, respectively) and exposed for 15 sec to
an acoustic signal of 10 ± 0.1 kHz frequency and 100 ± 1 dBA
intensity. This acoustic stimulus signal was produced by a signal
generator and projected via a high frequency speaker mounted on the
roof of the chamber. The noise level was measured close to the
animal’s ear by an external decibel-meter probe. Each animal was
subjected to a single audiogenic stimulation. Audiogenic seizures
were videotaped. The test measured the capacity of a test compound
to provide complete protection against threshold seizures induced
by 100 dBA. The duration of 4 successive phases [Latency, Wild
Running, Convulsions, Recovery] was recorded in seconds.
Statistical analysis. Data were expressed as mean ± SEM and
analysed by Student’s t-test.
Results
The body weight was similar in both groups after 30 days of the two
Mg-deficient diets: about 26 g (table
1). The individual spontaneous locomotor activity, measured
for 3 min (Apelex actimeter), showed that magnesium deficiency
induced central nervous hyperexcitability (NHE) in the
corn/sunflower group as compared to the rapeseed group (152.7 ±
37.9 vs. 97.0 ± 22.5). In the MES test, the mean intensity
responsible for a tonic seizure in 50% of the mice was slightly
higher in the rapeseed group (4.5 mA) than in the
corn/sunflower group (4 mA, NS). In addition, the rapeseed fed
mice recovered more rapidly (data not shown). In the MDDAS test,
the mice did not respond in the same way, depending on their diet:
(i) the number of convulsive mice was lower in the rapeseed group
(50%) as compared to the corn/sunflower group (100%). In addition,
all the mice convulsing in the rapeseed group recovered whereas 43%
died in the corn/sunflower group. (ii) The pattern of seizures was
also different. The first two phases of the audiogenic seizure test
increased significantly (p < 0.05) in the rapeseed group:
Latency and Wild running durations were 6.7 ± 5.5 and 3.7 ± 0.5 sec
instead of 4.0 ± 1.4 and 2.3 ± 0.4 sec respectively in the
corn/sunflower group, Convulsions and Recovery durations showed a
tendency to decrease slightly (table
2).
Table 1 Comparison of the two magnesium-deficient diets
on body weight gain, locomotor activity at the end of the
deprivation period (n = 14 per group).
|
Parameters Diets
|
Body Weight (g)
|
Locomotor activity
|
|
Corn/Sunflower (ALA deficient diet)
|
26.6 ± 0.88
|
152.7 ± 37.9
|
|
Rapeseed (ALA rich diet)
|
26.1 ± 1.18
|
97.0 ± 22.5*
|
Table 2 Comparison of the two magnesium-deficient diets
on the pattern of MDDAS test (n = 14 per group).
|
Diets
|
% of convulsing mice
|
Latency (sec)
|
Wild running (sec)
|
Convulsions (sec)
|
Recovery (sec)
|
|
Corn/sunflower (ALA deficient diet)
|
100
|
4.0 ± 1.4
|
2.3 ± 0.4
|
1.7 ± 0.4
|
46.5 ± 6.7
|
|
Rapeseed (ALA rich diet)
|
50
|
6.7 ± 5.5*
|
3.7 ± 0.5*
|
1.6 ± 0.5
|
43.3 ± 4.1
|
Discussion
Results reported in this work suggest a potential therapeutic value
of ALA for convulsive pathologies as previously proposed by others
in different animal seizure models [9, 2]. Firstly, magnesium
deficiency induces in Mg-deficient/ALA poor diet group a central
nervous hyperexcitability which did not appear in the
Mg-deficient/ALA rich diet group. Secondly, in the MES test, the
rapeseed fed group resisted slightly better to voltage stimulation
(4.5 mA) than corn/sunflower oil fed group (4mA), indicating
that sodium channel inhibition might be at least partly implicated
in the rapeseed protective effect. Interestingly, phenitoinergic
drugs (phenitoin, carbamazepine), which are the most used drugs in
preventing epilepsia, act on the same channels. Finally, in the
MDDAS test, the number of convulsive mice was significantly lower
in the rapeseed group (50% instead of 100% in the corn/sunflower
group) and the crisis was less severe since no mice died whereas
43% died in the other group. The global pattern showed an important
increase in the first step duration, suggesting as previously
voltage-dependent Na+ channel inhibition. But, the
global pattern would be rather GABAergic.
In any case, a rapeseed diet must not be considered as an
antiepileptic drug; but it may help to reduce the susceptibility to
epilepsia.
Interestingly, ALA has been shown to be neuroprotective both in
vivo (in kainate induced seizure test) and in vitro (on
seizure-like activity using glutamate neurons) and was associated
with blockade of glutamatergic transmission (6).
To conclude, these preliminary results suggest that chronic
dietary intake of rapeseed oil, an ALA rich monounsaturated oil,
could help to control neuronal disorders as here shown in our model
of magnesium-deficient mice.
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