The β2-adrenoceptor agonist clenbuterol elicits neuroprotective, anti-inflammatory and neurotrophic actions in the kainic acid model of excitotoxicity
Introduction
Excitotoxicity has been implicated as a major pathological mechanism underlying neuronal death in a range of acute and chronic neurodegenerative states including ischemia, traumatic brain and spinal cord injuries, epilepsy, Alzheimer’s disease and Parkinson’s disease (Hirose and Chan, 1993, Koutsilieri and Riederer, 2007, Meldrum, 1993, Park et al., 2004, Yi and Hazell, 2006). One model of excitotoxic cell death is the administration of the glutamate mimetic, kainic acid (KA), a potent agonist at the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and kainate subtypes of glutamate receptor (Wang et al., 2005). Systemic administration of KA induces a period of generalized seizures, followed by neuronal death which exhibits characteristics of apoptosis. Specifically, KA activates caspase-3; a key enzyme that drives the apoptotic process (Faherty et al., 1999), and induces DNA fragmentation in the CA3 and CA1 regions of the hippocampus consistent with the occurrence of apoptotic cell death (Lee et al., 2008). KA-induced neurotoxicity is associated with neuroinflammation characterized by increased expression of pro-inflammatory cytokines including IL-1β and TNF-α, and expression of inducible nitric oxide synthase (iNOS) (Jin et al., 2009, Lee et al., 2008, Wang et al., 2005). Furthermore, evidence indicates that inhibition of aspects of the neuroinflammatory response ameliorates KA-induced neurotoxicity (Chuang et al., 2007, Heo et al., 2006, Kunz and Oliw, 2001, Panegyres and Hughes, 1998, Tikka et al., 2001). In addition, other studies indicate that neurotrophic factors including BDNF and GDNF play a role in ameliorating KA-induced neurotoxicity (Khaspekov et al., 2004, Tandon et al., 1999, Yoo et al., 2006).
There is growing interest in central β2-adrenoceptors as a neuroprotective target due to the fact that stimulation of β2-adrenoceptors expressed on glial cells induces expression of neurotrophic factors, and also promotes an anti-inflammatory phenotype in glial cells (Counts and Mufson, 2010, Culmsee et al., 1999a, Culmsee et al., 1999b, Follesa and Mocchetti, 1993, Hertz et al., 2004, McNamee et al., 2010a, McNamee et al., 2010b, McNamee et al., 2010c). Clenbuterol is a brain penetrant β2-adrenoceptor agonist used in the treatment of respiratory disorders including asthma and chronic obstructive pulmonary disease (Baronti et al., 1980, Boner et al., 1988, Papiris et al., 1986), and has been shown to have neuroprotective properties both in vitro and in vivo (Culmsee et al., 1999a, Culmsee et al., 1999b). Specifically, clenbuterol has neuroprotective actions in rodent models of cerebral ischaemia (Junker et al., 2002, Culmsee et al., 1999b, Semkova et al., 1996, Zhu et al., 1998) and in an in vitro model of excitotoxicity, clenbuterol elicits protective effects by reducing apoptosis (Semkova et al., 1996). Data also indicate that clenbuterol has synergistic neuroprotective effects in a model of ischaemic stroke when administered in combination with the NMDA receptor antagonist-memantine (Culmsee et al., 2004). More recent studies demonstrate that clenbuterol has neuroprotective effects in a murine model of motor neurone disease and enhances cognition in aged rats (Ramos et al., 2008, Teng et al., 2006).
The aim of this study was to examine the ability of clenbuterol to promote production of neurotrophic factors and an anti-inflammatory phenotype in the hippocampus and to protect against KA-induced neurotoxicity.
Section snippets
Animals
Male Wistar rats (200–250 g) were obtained from the bioresources unit in TCD. Rats were maintained on a 12 h light:12 h dark cycle (lights on at 08:00 h) in a temperature controlled room (22 ± 2 °C) and food and water were available ad libitum. The experimental protocols were in compliance with the European Communities Council directive (86/609/EEC).
Drugs
Kainic acid (KA) was obtained from Ascent Scientific, UK and clenbuterol was obtained from Tocris Bioscience, UK. Both drugs were dissolved in 0.9% NaCl
Clenbuterol treatment does not alter KA-induced seizure behaviour
KA induced an increase in seizure behaviour which was not affected by pre-treatment with clenbuterol. Wet dog shakes, forelimb clonus with rearing and falling were observed in both treatment groups (Fig. 1). Data were analysed by repeated measures ANOVA but no differences between treatment groups were found. Total mean score ± SEM following KA treatment alone was 42 ± 0.6, and following clenbuterol pre-treatment was 42 ± 1.1.
Clenbuterol attenuated KA-induced apoptosis
KA treatment induced DNA fragmentation in the CA3 subfield of the
Discussion
Systemic injection of KA to rats induces progressive seizures and leads to neuronal death and inflammatory reaction in the hippocampus. The major finding in this study is that treatment with the brain penetrant β2-adrenoceptor agonist, clenbuterol, had a neuroprotective effect in the KA model of excitotoxicity indicated by its ability to reduce KA-induced DNA fragmentation in the CA3 region of the hippocampus, and to reduce hippocampal caspase-3 activation. These data complement previous
Acknowledgments
This research was supported by the Irish Research Council for Science Engineering and Technology and the Health Research Board of Ireland.
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