University of Ljubljana,
Medical Faculty, Institute of Pathophysiology,
Brain Research Laboratory, Slovenia
Abstract
Reversible acetylcholinesterase inhibitor
donepezil displays prophylactic effects against
intoxication with irreversible organophosphorous
acetylcholinesterase inhibitors. We used
behavioural observation of yawning and
epileptic seizures, histochemical
acetylcholinesterase staining, and in situ
hybridization of the immediate early genes,
c-fos and synaptotagmin 4 (Syt4) mRNAs in the
brain, to evaluate whether donepezil could
protect the brain against the effects of the
organophosphate anticholinesterase,
diisopropylfluorophosphate, in a rat model of
intoxication.
Diisopropylfluorophosphatetreated animals
exhibited frequent yawning, significant
inhibition of acetylcholinesterase staining and
upregulation of c-fos mRNA, but not the
epileptic seizures or significant change of Syt4
mRNA levels. In order to reduce the threshold
for the induction of cholinergic seizures,
additional groups of rats were pre-treated with
LiCl 24 h before the treatment with
diisopropylfluorophosphate. These rats exhibited
the seizures, a significant inhibition of
acetylcholinesterase staining and significant
upregulation of c-fos and Syt4 mRNA levels. All
the above-mentioned effects of
diisopropylfluorophosphate were inhibited by
donepezil pre-treatment. Donepezil pre-treatment
by itself induced only a comparatively weaker
inhibition of acetylcholinesterase staining and
infrequent yawning.
We conclude that donepezil protects the
brain against diisopropylfluorophosphate-induced
effects and that Syt4 mRNA upregulation may
serve as a novel marker for
organophosphate-induced seizures.
Introduction
Irreversible organophosphorous (UP)
inhibitors of acetyicholinesterase (AChE) were
developed as chemical warfare agents (e.g.,
soman) and agricultural insecticides (e.g.,
diisopropyl fluorophosphate - DFP). These
compounds exert their toxic effects by
(pseudo)irreversible inhibition of AChE,
resulting in prolonged acetylcholine (ACh)
activity, with subsequent excessive stimulation
of both muscarinic and nicotinic ACh receptors
(Taylor, 1996). Due to the localization of
cholinergic synapses in the central nervous
system (CNS), autonomic ganglia, and
neuromuscular junction, this leads to a
cholinergic toxic syndrome, characterized by
numerous symptoms of central and peripheral
origin. Since UP anticholinesterases are highly
soluble in lipid membranes, they easily cross
the blood-brain-barrier (BBB). The spectrum of
acute CNS effects includes confusion, ataxia,
slurred speech, loss of reflexes, Cheyene-Stokes
respiration, coma, and central respiratory
paralysis (Watson et al., 2009). Cholinergic
mechanisms can also trigger the onset of
seizures (Turski et al., 1983), although the
propagation and maintenance of status
epilepticus occurs primarily via activation of
excessive glutamatergic transmission (Smolders
et al., 1997) and subsequent excitotoxic brain
damage (Solberg and Belkin, 1997). Conventional
treatment of organophosphate poisoning includes
combined administration of a cholinesterase
reactivator (an oxime), a muscarinic cholinergic
receptor antagonist (atropine) and a
benzodiazepine anticonvulsant (diazepam) (Watson
et al., 2009).
One of the strategies for prevention of
irreversible inhibition of AChE by UP is the use
of reversible inhibitors, such as carbamates
(Lallement et al., 2001). AChE inhibited by
carbamates is resistant against irreversible
inhibition by UP nerve agents and the reversibly
inhibited enzyme recovers spontaneously, without
the use of AChE reactivators. The peripherally
acting carbamate AChE inhibitor, pyridostigmine
bromide (PB), has been advocated as a
prophylaxis against potential threat with OP
warfare agents (Keeler et al., 1991). However,
PB does not cross the BBB and thus may not
counteract CNS symptoms. Indeed, the CNS effects
of OP agents may actually be potentiated by
pre-treatment with PB (Amourette et al., 2009).
In this regard, centrally acting reversible
anticholinesterases that have been used for the
treatment of Alzheimer's disease (AD) may offer
a better prophylactic choice against OP
intoxication than PB.
Donepezil (DON) is a reversible, primarily
non-competitive, selective inhibitor of AChE
used for the treatment of AD. Acute
pre-treatment with oral DON, with and without
scopolamine, decreased the hypothermic,
hypokinetic, and diarrhoea-inducing effects of
DFP (Janowsky et al., 2004, 2005). A combined
pre-treatment with DON and procyclidine, a
muscarinic receptor antagonist, protected
against soman-induced seizures (Haug et al.,
2007).
Here, we further explored the prophylactic
potential of DON against the central effects of
DFP in the brain, such as yawning
(Ogura
et al., 2001) and seizures (Zivin et al., 1999).
In addition, we performed AChE histochemical
staining and in situ hybridization of immediate
early genes (lEGs) c-fos and synaptotagmin 4
(Syt4) mRNA for this purpose. c-fos induction in
the brain is a well-established marker of
neuronal hyperactivity induced by cholinergic
seizures (Zivin et al., 1999), while there is
rising evidence for the pathophysiological and
adaptive changes of membrane trafficking in the
brain induced by seizures, thus implicating the
potential role of the plasticity of Syt4
expression in these processes (Glavan et al.,
2009).
Discussion
The main findings of our experiments
confirmed that DON, which is the dominant drug
in the treatment of Alzheimer's disease, could
also display prophylactic activity against
central signs of intoxication with OP nerve
agents.
AChE staining showed that DFP almost fully
inhibited striatal, cortical and hippocampal
AChE. Although to a lesser extent, DON also
inhibited AChE in these regions. However, it
should be remembered that DFP induces
irreversible inhibition of AChE, while the
inhibition of AChE by DON is mostly reversible.
The only physiological mechanism for restoring
DFP-inhibited AChE activity thus depends on the
relatively slow process of de novo synthesis of
AChE. It may be speculated that the
pre-treatment with DON has protected AChE
against the irreversible inhibition by DFP, and
that the relatively high AChE activity observed
in our experiments 1-4 h after the treatment
with DFP reflects spontaneously recovered
activity of AChE.
In rats without seizures, we did not find
cortical and hippocampal c-fos mRNA
up-regulation after DON, as compared to the
clear up-regulation in DFP-treated animals. DON
in fact completely prevented the DFP-induced
up-regulation of c-fos mRNA levels. lEGs such as
c-fos respond to various stimuli and their
protein products transactivate other genes,
resulting in longterm changes in the nervous
system. Various types of noxious stimulation
(e.g., thermal, mechanical, and chemical),
including DFP, have been shown to induce c-fos
in the brain and spinal cord of various species
(Zivin et al., 1999; Gupta et al., 2000). Since
the massive induction of c-fos mRNA is
indicative of pathologic processes, such as
excitotoxic and neurodegenerative changes (Gupta
et al., 2000), the prevention of DFP-induced
upregulation of c-fos mRNA indicates that
prophylaxis with DON may prevent
neurodegenerative changes induced by DFR Syt4 is
also considered to be an lEG, since its
induction does not depend on previous protein
synthesis (Vician et al., 1995). However, Syt4
does not seem to be involved in the regulation
of delayed gene response. Syt4 has rather been
recognized to be implicated in phenotypic
changes involving membrane trafficking (Glavan
et al., 2009). Syt4 mRNA levels were not
significantly affected by either DON or DFP.
This suggests that in rats without seizures, the
membrane trafficking may not be significantly
affected by the cholinergic stimulation and that
the increase of c-fos mRNA levels is a more
rapid and sensitive marker for increased brain
cholinergic activity.
So far, no data exist in the literature in
regard to the prophylactic effects of DON
against DFP-induced seizures. Syt4 mRNA is known
to be strongly up-regulated in the brains of
animals with seizures induced with glutamate
analogue kainic acid (Glisovic et al., 2007).
There is rising evidence for the
pathophysiological and adaptive changes of
membrane trafficking in neurodegeneration in
neuronal plasticity and in glial activation
induced by seizures, thus implicating the
potential role of the plasticity of Syt4
expression in these processes.
So far, Syt4 mRNA levels (Tocco et al.,
1996) and protein (Glisovic et al., 2007) were
found to be up-regulated in the brain regions
associated with the propagation of temporal lobe
epilepsy only following kainic acid-induced
seizures, and in hemi-seizures in rats after
unilateral striatal injection of glutamate
analogue and excitotoxin quinolinic acid (Glavan
et al., 2009). Numerous studies have
demonstrated that excitatory amino acid
glutamate also plays a prominent role in the
maintenance of organophosphate-induced seizures
and in the subsequent neuropathology especially
through over-activation of the
N-methyl-D-aspartate (NMDA) receptor subtype
(Lallement et al., 1999). We have shown
previously that prevention of hemi-seizures in
rats after unilateral striatal injection of
glutamatergic NMDA agonist quinolinic acid by
pre-treatment with NMDA antagonist dizolcipine
(MK-801) prevented up-regulation of c-fos and
Syt4 mRNAs (Glavan et al., 2009). In the present
experiment, we also found that the prerequisite
for the up-regulation of Syt4 mRNA was the
glutamatergic hyperactivity due to the induction
of seizures, and not just cholinergic
hyperactivity by itself. The increase of
cholinergic activity in DON-treated rats without
seizures or in rats in which LiCIIDFP-induced
seizures were prevented by DON namely did not
result in increased Syt4 mRNA levels. It is
noteworthy in this regard that DON has been
shown to protect cortical neurons against
glutamate-induced neurotoxicity and apoptotic
death via nicotinic ACh receptors (Takada et
al., 2003). It remains to be determined whether
this may be an additional pharmacological
mechanism by which DON could prevent the
up-regulation of Syt4 mRNA in animals with
seizures.
We conclude that the pre-treatment with DON
protects the brain against the effects of
central cholinergic and glutamatergic
over-activity induced by DFP. This study has
also revealed that up-regulation of Syt4 mRNA
may be considered as a novel marker for
increased membrane trafficking in animals with
OP-induced seizures.
-Ogura
H, Kosasa T, Kuriya Y, Yamanishi Y Central
and peripheral activity of cholinesterase
inhibitors as revealed by yawning and
fasciculation in rats. Eur J Pharmacol.
2001;415(2-3):157-64
