The β₂-adrenoceptor agonist clenbuterol elicits neuroprotective, anti-inflammatory and neurotrophic actions in the kainic acid model of excitotoxicity

Abstract

Excitotoxicity is a mechanism of neuronal cell death implicated in a range of neurodegenerative conditions. Systemic administration of the excitotoxin kainic acid (KA) induces inflammation and apoptosis in the hippocampus, resulting in neuronal loss. Evidence indicates that stimulation of glial β₂-adrenoceptors has anti-inflammatory and neurotrophic properties that could result in neuroprotection. Consequently, in this study we examined the effect of the β₂-adrenoceptor agonist clenbuterol on KA-induced inflammation, neurotrophic factor expression and apoptosis in the hippocampus. Clenbuterol (0.5 mg/kg) was administered to rats one hour prior to KA (10 mg/kg). Epileptic behaviour induced by KA was assessed for three hours following administration using the Racine scale. Twenty-four hours later TUNEL staining in the CA3 hippocampal subfield and hippocampal caspase-3 activity was assessed to measure KA-induced apoptosis. In addition, expression of inflammatory cytokines (IL-1β and IFN-γ), inducible nitric oxide synthase (iNOS), kynurenine pathway enzymes indolamine 2,3-dioxygenase (IDO) and kynurenine monooxygenase (KMO), the microglial activation marker CD11b, and the neurotrophins BDNF and NGF were quantified in the hippocampus using real-time PCR. Whilst clenbuterol treatment did not significantly alter KA-induced epileptic behavior it ameliorated KA-induced apoptosis, and this neuroprotective effect was accompanied by reduced inflammatory cytokine expression, reduced expression of iNOS, IDO, KMO and CD11b, coupled with increased BDNF and NGF expression in KA-treated rats. In conclusion, the β₂-adrenoceptor agonist clenbuterol has anti-inflammatory and neurotrophic actions and elicits a neuroprotective effect in the KA model of neurodegeneration.

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.