Metab Brain Dis (2009) 24:453–461 DOI 10.1007/s11011-009-9144-7 O R I G I N A L PA P E R
Basal ganglia neuroprotection with anticonvulsants after energy stress: a comparative study S. Arpin & E. Lagrue & S. Bodard & S. Chalon & P. Castelnau
Received: 23 April 2009 / Accepted: 9 July 2009 / Published online: 30 September 2009 # Springer Science + Business Media, LLC 2009
Abstract The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model provides a valuable paradigm of the energy deficiency disorders found in childhood. In such disorders, anticonvulsants may provide neuroprotection by modulating cellular energy consumption and by exerting favorable pleiotropic effects on neuronal survival. To verify such hypothesis, we tested the effects of levetiracetam, vigabatrin, gabapentine, pregabaline, tiagabine, clonazepam and lamotrigine on neuroprotection in the MPTP mouse model. The membrane dopamine transporter (DAT) density, which provides a reliable index of dopaminergic neurons survival in the basal ganglia, was assessed by semi-quantitative autoradiography of the striatum. Unlike all other anticonvulsants tested, lamotrigine provided a significant and dosedependent neuroprotection in these experimental conditions. Lamotrigine, a widely used and well-tolerated molecule in children, could provide neuroprotection in various energy deficiency disorders. Keywords Basal ganglia . Mitochondria . MPTP . Leigh syndrome . Neuroprotection . Anticonvulsant S. Arpin : E. Lagrue : S. Bodard : S. Chalon : P. Castelnau UMRS INSERM U 930, CNRS ERL 3106, “Imagerie et cerveau”, Tours 37000, France S. Arpin : E. Lagrue : S. Chalon : P. Castelnau Université François Rabelais de Tours, Tours 37000, France E. Lagrue : P. Castelnau CHRU de Tours, Pôle de Pédiatrie Médicale, Unité de Neuropédiatrie, Hôpital Clocheville, Tours 37000, France P. Castelnau (*) Unité de Neuropédiatrie et UMRS INSERM U 930, CNRS ERL 3106, “Imagerie et cerveau”, Université François Rabelais Laboratoire de Biophysique Médicale et Pharmaceutique, 31 Avenue Monge, Tours 37200, France e-mail:
[email protected]
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Introduction Evidence from degenerative and metabolic encephalopathy studies indicate that the substantia nigra (Chalon et al. 1999) and the striatum are vulnerable structures in the brain, especially during brain development (Groenendaal et al. 2006; Saudubray and Charpentier 1995). A partial or total destruction of this nigro-striatal dopaminergic network, involved in multiple motor, sensory, and cognitive functions, is found in several conditions occurring in childhood such as various mitochondrial energy production deficiencies including Leigh syndrome (Cavanagh and Harding 1994; Saudubray and Charpentier 1995). These basal ganglia share a common susceptibility to energetic stress (Beal 2000; Browne et al. 1997), which can be studied experimentally using mitochondrial inhibitors such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Schulz et al. 1996). In both animals and humans, this toxin depletes the dopaminergic neuron bodies located in the SN, causing the degeneration of the dendrite arborescence projected into the striatum (Przedborski et al. 2000). In a previous study, we showed that MPTP intoxication in young mice, which leads to energy-dependent deterioration of the nigro-striatal pathway, provides a valuable pharmacological model of Leigh syndrome and other mitochondrial disorders in humans (Lagrue et al. 2009). This model opens new avenues of neuroprotective approaches for mitochondrial disorders and energy stress, including during development. Various neuroprotection strategies have been tested in the MPTP mouse model, including energy sparing drugs, free radicals scavengers, anti-glutamatergic drugs, anti-apoptotic molecules, and neurotrophins (Alexi et al. 2000; Matthews et al. 1999; Srivastava et al. 1993). In the present study we evaluated the neuroprotective effects of anticonvulsants because they favorably modulate a large number of cellular metabolic pathways; indeed, anticonvulsants induce glutamate release inhibition, GABAergic stimulation, and ion channel inhibition. These combined pharmacological properties promote the stabilization of neuronal plasma membranes and the reduction of ATP-dependent ion channel activity. This might subsequently protect neurons through an adaptation of the cellular energy consumption to the limited ATP availability occurring during an energy stress (Obrenovitch and Urenjak 1998). Moreover, the current anticonvulsants have an acceptable tolerance profile in humans including young children. Therefore, anticonvulsants provide good candidates for neuroprotection, especially in energy stress conditions in childhood. Therefore, we evaluated various anticonvulsants used in pediatric clinical practice to determine their neuroprotective properties in such energetic disorders. We evaluated levetiracetam (LVT), vigabatrin (VGT), gabapentine (GPT), tiagabine (TGB), pregabaline (PGB), clonazepam (CZP), and lamotrigine (LMT). LVT, is a relatively new anticonvulsant, which provides an interesting protection against mitochondrial dysfunction in the hippocampus after status epilepticus (Gibbs et al. 2006). VGT, GPT, and TGB exhibit neuroprotective properties which have been reported in other pathological conditions such as status epilepticus and hypoxia-ischemia (Andre et al. 2001; Iqbal et al. 2002; Williams et al. 2006). PGB and CZP have never been tested for neuroprotection. To our knowledge, none of these anticonvulsants has been tested in the MPTP mouse model, except LMT which has neuroprotective properties when combined with MPTP administration (Jones-Humble et al. 1994; Lagrue et al. 2009, 2007; Schulz et al. 1996) through yet unclear mechanisms. All anticonvulsants
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which could modify MPTP toxicity through cytochrome P450 or monoamine oxidase B activation (i.e., carbamazepine, phenytoin, phenobarbital, primidone, stiripentol, valproic acid, topiramate, zonisamide, oxcarbazepine, felbamate, and ethosuximide) were excluded (Przedborski et al. 2000). In the present study, we compared the potential neuroprotective properties of seven anticonvulsants on MPTP-intoxicated mice by measuring the density of the dopamine transporter (DAT), a validated highly sensitive and specific dopaminergic marker of the nigro-striatal network.
Material and methods All experiments were performed on consanguineous male C57/Bl6N@Rj mice (5 weeks old, average weight: 19±1 g, CERJ, France). The mice were kept under controlled conditions on a 12-hour light/dark cycle with food and water ad libitum in accordance with the European Community Commission guidelines (86/609/EEC). Eleven groups (n=6 per group) were constituted: control (saline per os [p.o.] and intraperitoneally [i.p.]), MPTP (saline p.o. and MPTP i.p.), GPT-MPTP (GPT p.o. and MPTP i.p.), CZP-MPTP (CZP p.o. and MPTP i.p.), VGT-MPTP (VGT p.o. and MPTP i.p.), LVT-MPTP (LVT p.o. and MPTP i.p.), PGB-MPTP (PGB p.o. and MPTP i.p.), TGB-MPTP (TGB p.o. and MPTP i.p.), and LMT-MPTP (LMT p.o. and MPTP i.p.). Each group was treated with the corresponding anticonvulsant twice per day p.o. from Day 1 (D1) to Day 7 (D7). The cumulative dose were 50 mg/kg/day for GPT (Pfizer, France), 0.1 mg/kg/day for CZP (Roche, France), 200 mg/kg/day for VGT (Marion Merrell, France), 1,000 mg/kg/day for LVT (UCB, France), 10 mg/kg/day for PGB (Pfizer, France), 17 mg/kg/day for TGB (Cephalon, France) and 40 mg/kg/day for LMT (GlaxoSmithKline, France). Anticonvulsant doses were chosen based on previous reports of neuroprotective effect for the drug or, when missing, extrapolated from the dosage used in humans as anticonvulsant (Jones-Humble et al. 1994; Lagrue et al. 2009, 2007; Schulz et al. 1996). Two additional dosages of 5 and 20 mg/kg/day (LMT5 and LMT20) were tested in further experiments with LMT. Each anticonvulsant was dissolved in saline to obtain a final 200 µL volume of oral administration. MPTP (Sigma, Paris, France) was dissolved in 0.9% sodium chloride to a final concentration of 2.5 mg/mL. Mice were intoxicated with four administrations of MPTP (12.5 mg/kg; 100 µL injection per 20 g of mouse body weight) i.p. at 1-hour intervals on Day 3. Each mouse was weighted daily. The mice were sacrificed by cervical dislocation 7 days after MPTP intoxication (i.e., 3 days after the end of the anticonvulsant treatment). The brains were rapidly removed, weighted, frozen (at −35°C), subsequently cut into 20-µm coronal sections (Reichert-Jung Cryocut CM3000 Leica, France), and stored at −80°C. To measure the level of DAT density in the dopaminergic neuron dendrites projected into the striatum, semi-quantitative autoradiographic binding studies were performed using the specific radio-labeled ligand [125I]PE2I as previously described (21). Briefly, 3 sections per animal were incubated for 90 min at room temperature (RT) in a phosphate buffer containing [125I]PE2I (100 pM). Non-specific binding was assessed on adjacent sections with the same buffer containing 1 µM cocaine
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(Cooper, France), and subtracted from total binding to yield the specific binding. After rinsing and drying, the sections were exposed to sensitive films (Biomax MR, Kodak, France) with radioactive standard scales (125I-microscales, Amersham Bioscience AB, UK). The films were analyzed through an image analyzer (Betavision, Biospace Instruments, France) after identifying the striatum areas. The absorbance obtained was converted into apparent tissue ligand concentrations with reference to standards and specific activity of the radioligand (74 TBq/mmol). The intensity of [125I]PE2I binding was expressed in Bq/mg of equivalent tissue (mean ± standard deviation [SD]). The data were prospectively collected and analyzed using the Winks software (Texasoft, USA). Statistical significance was evaluated with the non parametric Mann-Whitney test for non-paired values from small groups. Statistical significance was considered for P
