American Journal of Animal and Veterinary Sciences 6 (4): 166-170, 2011 ISSN 1557-4555 2011 Science Publications
Anticonvulsant Effect of Pitavastatin in Mice Induced by Pentylenetetrazole
Department of Pharmacology and Toxicology, Faculty of Veterinary,
Tabriz Branch, Islamic Azad University, Tabriz, Iran
Abstract: Problem statement: The 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) have been unequivocally shown to reduce cardiovascular morbidity and mortality by their lipid-lowering actions. It has been suggested that statins appear to have therapeutic benefits in diseases that are unrelated to elevated serum cholesterol levels. The aim of this study was to evaluation the effect of intraperitoneally injection of different doses of pitavastatin on PTZ-induced seizure threshold in mice. Approach: Mice were divided into 9 groups randomly: the first group received saline normal (ip) (control group); the second group received Carboxymethylcellulose (CMC) 0.5% (ip) (vehicle group) and the next groups received respectively different doses of pitavastatin (1, 5, 10,
20, 40, 80 and 100 mg kg daily ) for 4 days (ip) before determination of seizure threshold induced by PTZ. Results: Results showed that PTZ-induced seizure threshold in control mice was 35.48±1.39 mg
kg . Intraperitoneal injection of pitavastatin showed significant (p<0.05) increase of PTZ-induced seizure threshold in a dose dependently manner. Conclusion: According to our results, pitavastatin has anticonvulsant effects on mice. Nevertheless, new studies must be carried out in order to determine the beneficial effects of statins in treatment of epilepsy. Key words: Threshold, Franeyltransferase Inhibitor (FTI), neurological diseases, PTZ-induced
seizure, elevated serum, lipid-lowering, control group, seizure threshold
dysfunction, anti-thrombosis and anti-oxidant actions,
prevention of Alzheimer’s disease and antineoplasic
Epilepsy is one of the major neurological diseases
actions (Goncalves et al., 2011). Statins might exert
in humans and about one percent of the population is
beneficial effects beyond cholesterol reduction; include
improving endothelial function, decreasing vascular
(HMG-CoA) reductase inhibitors (statins) have been
inflammation, inhibiting smooth-muscle proliferation
and immunomodulation. Most of these effects are
morbidity and mortality (NIH, 1994). Statins are
mediated through inhibition of isoprenoid synthesis, with
among the most widely used prescription drugs and
subsequent effects on multiple downstream signaling
exert their lipid-lowering actions by reversible and
pathways (Wang et al., 2008). Also statins might exert
competitive inhibition of the enzyme 3-hydroxy-3-
beneficial neuroprotective effects in various animal
disease models and clinical studies (Rangel et al.,
reductase), the rate-limiting step in the conversion of
2005). Furthermore, recent studies have shown that
3-Hydroxy-3-Methylglutaryl-coenzyme A (HMG-
statins reduces the extent of brain damage after
CoA) to mevalonate, a precursor of cholesterol
ischemic insult (Sironi et al., 2003). Therefore the aim
(McTaggart et al., 2001). It has been suggested that
of this study was to evaluation the effect of
statins appear to have therapeutic benefits in diseases
intraperitoneally injection of different doses of
that are unrelated to elevated serum cholesterol
pitavastatin on PTZ-induced seizure threshold in mice.
levels, such as rheumatologic diseases and ischemic
stroke (McCarey et al., 2004). Statins are also known
MATERIALS AND METHODS
for their pleiotropic effects, which are independent of
their lipid-lowering properties. Among the effects of
Animals: Experiments were performed on 22-25 g
statins, the most relevant are anti atherosclerotic and
adult NMRI male mice in their 8-9 week (n = 8 for each
anti-inflammatory actions, improvement of endothelial
group), purchased from Razi Institute (Iran). Animals
American J. Animal & Vet. Sci., 6 (4): 166-170, 2011
were housed 8 per cage in the Animal House of Veterinary Faculty of Tabriz Branch, Islamic Azad University in a temperature (20-22°C) and humidity (50±10%) controlled environment under a 12-h light
dark cycle (lights on at 7 am). Food and water were available ad libitum. This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Research affairs of Tabriz University of Medical Sciences, Tabriz-Iran. All efforts were made to minimize the number of animals which
were used and their suffering degree. Animals were
divided into 9 groups randomly: the first group received
Fig. 1: PTZ-induced seizure threshold in mice (mg/kg).
saline normal (ip) (control group); the second group
Effect of intraperitoneally injection of different
received Carboxymethylcellulose (CMC) 0.5% (ip)
doses of pitavastatin on seizure threshold. Each
(vehicle group) and the next groups received
respectively different doses of pitavastatin (1, 5, 10, 20,
*P<0.05, **P<0.01 and ***P<0.001 compared
40, 80 and 100 mg kg daily ) for 4 days (ip) before
DISCUSSION Chemicals: PTZ (Pentylenetetrazole) was purchased
from Sigma-Aldrich. Pitavastatin was provided from
Pentylenetetrazole (PTZ) has been used widely to
Kowa Pharmaceuticals. All other reagents were of
produce the animal model of chemically induced
analytical grade. Pitavastatin was prepared by being
seizure because this model is highly sensitivity for
dissolved in 0.5% Carboxymethylcellulose (CMC) and the
comparing different chemical under standardized
vehicle group was given an equal volume of vehicle.
conditions (Shafaroodi et al., 2004). In this study PTZ-
induced seizure threshold in control mice was
PTZ-induced clonic seizure threshold: Behavioral
35.48±1.39 mg kg and pitavastatin increased PTZ-
experiments were done in a quiet, temperature-
induced seizure threshold in a dose dependently
controlled (20-22°C) room between 10 am and 4 pm.
manner. Anti-nociceptive and anti-inflammatory effects
PTZ-induced clonic seizure threshold was determined
of statins have been shown in different experimental
by inserting a 30-gauge needle into the tail vein of mice
animal models of pain and inflammatory and clinical
and infusion of 0.5% PTZ solution at a constant rate of
studies (Dantas et al., 2010; Massaro et al., 2010; Nezic
1 ml/min to unrestrained freely moving animals.
et al., 2009; Sparrow et al., 2001; Weitz-Schmidt et al.,
Minimal dose of PTZ (mg kg ) needed to induce
2001), but so fare effects of statins on epilepsy are very
forelimb clonus followed by full clonus of the body was
limited (Rangel et al., 2005; Lee et al., 2008). Results
recorded as an index of clonic seizure threshold
of this study showed that pitavastatin has beneficial
neuroprotective effects on PTZ seizure model in mice
Data analysis: Group data are presented as mean±
that in agreement with a recent study that reported an
SEM and analyzed statistically using student test. Time
effective neuroprotective action of statins after an acute
course data were analyzed using one-way ANOVA
brain injury and epilepsy (Daimon et al., 2004; Lee et
followed by Tukey’s post hoc test. The level for
al., 2008; Rangel et al., 2005). Furthermore, recent
statistical significance was set at a p<0.05.
studies have shown that lovastatin partially suppressed
acute experimental autoimmune Encephalomyelitis
(EAE) in rat spinal cord injury model (26). Also,
studies reported that statin treatment provided anti-
PTZ-induced seizure threshold in control mice was
inflammatory effects on human multiple sclerosis and
35.48±1.39 mg kg . In vehicle group, CMC have not
X-linked adrenoleukodystrophy (Nath et al., 2004;
shown significant change on PTZ-induced seizure
Paintlia et al., 2005; Pannu et al., 2007; Shepherd et al.,
threshold compared with control group (Fig. 1).
1995; Singh et al., 1998; Stanislaus et al., 2002; 1999;
2001; Vollmer et al., 2004). Moreover, other research
significant (p<0.05) increase of PTZ-induced seizure
groups have reported the similar neuroprotective role of
threshold in a dose dependently manner compared with
statins in other neurological disease models, such as
traumatic brain injury, brain ischemia and Alzheimer’s
American J. Animal & Vet. Sci., 6 (4): 166-170, 2011
disease (Endres et al., 1998; Kirsch et al., 2003; Lu et
thus, their biological roles are expected to be mediated
al., 2004). Rangel et al. (2005) demonstrated that
by their inhibitory roles in cholesterol and isoprenoid
lovastatin treatment was able to prevent hippocampal
synthesis. Indeed, the inhibitory role of statins in
neuronal loss in CA1 subfield after an epileptic insult
cholesterol biosynthesis previously reported to inhibit
(Rangel et al., 2005). The molecular mechanisms of the
NMDA mediated excitotoxicity in vitro neuron culture
anticonvulsant and immunomodulatory properties of
study. Also has been reported that statin treatment of
statins have not been fully clarified. Rangel et al.
cultured neuron cells leads to instability of AMPA
(2005) suggested that Reactive Oxygen Species (ROS)
receptor by disruption of cholesterol and sphingolipid
are a part of normal human metabolism; however, when
rich membrane microdomains called “Detergent
produced in excess, ROS can cause tissue injury
Resistant/Insoluble Membrane domains (DRM/DIM)”
including lipid peroxidation, DNA damage and enzyme
or “lipid rafts”. Therefore, they had suggested that the
inactivation. ROS is a common denominator among
possible involvement of cholesterol biosynthesis in
acute neurological conditions, including epilepsy. In the
statin-mediated anti-excitotoxic activity (Lee et al.,
pilocarpine model, there is an involvement of
2008). At present, whether cholesterol biosynthesis
excitotoxic neuronal injury and ROS production has
plays a role in statin-mediated anti-seizure and anti-
been considered to be a part of mechanisms involved
excitotoxicity in the in vivo animal model is not known.
with glutamatergic excitotoxicity in vitro and in vivo.
In the brain, cholesterol has a very long half life (up to
Moreover, it was demonstrated that lovastatin treatment
several months). Therefore, statins are not expected to
inhibits free radical injury. Thus, this antioxidant effect
change brain cholesterol level readily. Previous studies
of statins could explain the neuroprotective properties
have reported that systemic statin treatment does not
found in their study (Rangel et al., 2005). Also, they
affect cholesterol levels in the brain of guinea pigs.
had been suggested that there is one classical argument
supporting a possible role of Nitric Oxide (NO) in
cholesterol levels in lipid raft fractions of synaptosomal
convulsive phenomena: excitatory amino acids, such as
plasma membrane suggests that statin treatment may
N-Methyl-D-Aspartate (NMDA) and kainate, are
affect intercellular or intracellular distribution of
known to be potent convulsants and the activation of
cholesterol which is associated with glutamate receptor
NMDA receptors is accompanied by the formation of
function in induction of status epilepticus and
NO. In fact, the role of NO in epileptogenesis has been
excitotoxicity (Lee et al., 2008). Along with
examined in a number of studies, suggesting being a
proconvulsive endogenous substance. Furthermore,
pyrophosphate and geranylgeranylpyrophosphate are
some studies have revealed that statins inhibit the
lipid byproducts produced by the mevalonate pathway
production of NO in brain parenchyma, indicating that
and implicated in membrane attachment and function of
statins, secondarily, may play the role of an
small GTPases. Since the small GTPases (i.e., RhoA
and H-Ras) are involved in various neuronal functions
glutamate-mediated neurotoxicity. Other possibility is
and excitotoxic signaling cascades, statins are also
that the inhibition of brain endothelial Nitric Oxide
expected to exert their anti-seizure and anti-excitotoxic
Synthase (eNOS) leads to increased blood pressure,
activities through inhibition of isoprenoid synthesis and
which, in turn, may affect the excitability of central
interfering with small GTPase signaling. Recent in vivo
nervous system; however, it was demonstrated that
reports have demonstrated that inhibition of H-Ras
statins are able to upregulate eNOS, may be pivotal in
farnesylation by Franeyltransferase Inhibitor (FTI)
treatment inhibits NMDA-mediated excitotoxicity in
decreasing with this, the firing threshold (Rangel et al.,
the rat brain. This report supports that statins may
2005). Also, they had been suggested that anti-
inflammatory effects of statins could also contribute to
isoprenylation. However, whether statins exert their
epilepticus. Moreover, several studies have implicated a
number of cytokines in seizure-related pathology.
inhibiting synthesis of isoprenoid or cholesterol or both
Therefore lovastatin treatment may provide an
of them is not known at present (Lee et al., 2008). Also,
important approach to suppression of the inflammatory
Lee et al. (2008) reported that neuroprotective
responses after status epilepticus (Rangel et al., 2005).
efficacies of statins are mediated by their anti-
In other study, Lee et al. (2008) reported that statins are
inflammatory activity under various neurological
inhibitors of HMG-CoA reductase, a rate limiting
disease conditions such as human multiple sclerosis and
enzyme for synthesis of cholesterol and isoprenoids,
its animal models, Spinal cord injury, X-linked
American J. Animal & Vet. Sci., 6 (4): 166-170, 2011
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