Elsevier

Brain Research

Volume 1390, 16 May 2011, Pages 126-141
Brain Research

Research Report
Administration of 2-arachidonoylglycerol ameliorates both acute and chronic experimental autoimmune encephalomyelitis

https://doi.org/10.1016/j.brainres.2011.03.020Get rights and content

Abstract

Background and purpose

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis (MS) and both conditions have been reported to exhibit reduced endocannabinoid activity. The purpose of this study was to address the effect of exogenously administered 2-arachidonoylglycerol (2AG), an endocannabinoid receptor ligand, on acute phase and chronic disability in EAE.

Experimental approach

Acute and chronic EAE models were induced in susceptible mice and 2AG-treatment was applied for 14 days from day of disease induction.

Key results

2AG-treatment ameliorated acute phase of disease with delay of disease onset in both EAE models and reduced disease mortality and long-term (70 days post-induction) clinical disability in chronic EAE. Reduced axonal pathology in the chronic EAE- (p < 0.0001) and increased activation and ramification of microglia in the 2AG-treated acute EAE- (p < 0.05) model were noticed. The latter was accompanied by a 2- to 4-fold increase of the M2-macrophages in the perivascular infiltrations (p < 0.001) of the 2AG-treated animals in the acute (day 22), although not the chronic (day 70), EAE model. Expression of cannabinoid receptors 1 (CB1R) and 2 (CB2R) was increased in 2AG-treated animals of acute EAE vs. controls (p < 0.05). In addition, ex vivo viability assays exhibited reduced proliferation of activated lymph node cells when extracted from 2AG-treated EAE animals, whereas a dose-dependent response of activated lymphocytes to 2AG-treatment in vitro was noticed.

Conclusion and implications

Our data indicate for the first time that 2AG treatment may provide direct (via CBRs) and immune (via M2 macrophages) mediated neuroprotection in EAE.

Research Highlights

► Exogenous 2AG-treatment is applied for the first time in PLP- and MOG-induced EAE. ► 2AG delayed disease onset and reduced relapse severity and long-term disability in EAE. ► 2AG inhibited lymphocyte-activation and induced apoptosis. ► 2AG-treatment reduced axonal pathology in the chronic EAE model. ► 2AG-induced neuroprotection may be microglia-related in acute EAE.

Introduction

Experimental autoimmune encephalomyelitis (EAE) is an experimental model of immune-mediated demyelination and axonopathy within the central nervous system (CNS) of susceptible animals, using the triggering of various myelin antigens (Grigoriadis et al., 2004, Grigoriadis et al., 2006).

Among the several factors that play a role in the pathogenesis of both EAE and MS is the endocannabinoid (eCB) system (Centonze et al., 2007a, Shohami and Mechoulam, 2006, Witting et al., 2006) which includes the endogenous cannabinoids (eCB) N-arachidonoylethanolamine (anandamide) (Devane et al., 1992) and 2-arachidonoylglycerol (2AG) (Mechoulam et al., 1995, Sugiura et al., 1995). Both compounds act via the CB1 and CB2 receptors (CB1R and CB2R, respectively) (Matsuda et al., 1990, Munro et al., 1993) or the recently described non-CB receptors (De Petrocellis and Di Marzo, 2010). The CB1R are expressed mainly in the brain, pituitary gland, immune cells, and reproductive tissues, whereas CB2R are found primarily on immune cells and brain as well (Onaivi et al., 2006). Anandamide binds with higher affinity to the brain CB1R than CB2R, whereas 2AG exhibits similar affinity for both receptors (Devane et al., 1992, Pandey et al., 2009). 2AG has been suggested to be the main natural ligand of both central (neural) and peripheral (immune) cannabinoid receptors (Berdyshev, 2000, Gonsiorek et al., 2000, Sugiura et al., 2000, Sugiura et al., 2004) with multiple effects on lymphocytes (Lee et al., 1995, Ouyang et al., 1998, Rockwell et al., 2006), microglial cells (Carrier et al., 2004), and macrophages function (Gallily et al., 2000). Based on these data, it has been assumed that 2AG could be a potent modulator of immune-mediated CNS diseases (Centonze et al., 2007b, Consroe, 1998).

Various pathological conditions increase the “eCB tone” (Franklin et al., 2003, Hashimotodani et al., 2007, Witting et al., 2004b, Witting et al., 2006), and it has been postulated that this response is neuroprotective (Franklin et al., 2003, Guzman et al., 2001, Mechoulam and Shohami, 2007, Nagayama et al., 1999, Panikashvili et al., 2001, Wallace et al., 2003). However, MS and EAE render a “dysregulation” of brain eCB response (Centonze et al., 2007a, Shohami and Mechoulam, 2006, Witting et al., 2006) and therefore constitute an exception from the “rule” of increased eCB tone noticed in other neuropathologies (Franklin et al., 2003, Panikashvili et al., 2001, Witting et al., 2004b).

Whatever the cause for this discrepancy, the rationale for interference with exogenously administered 2AG, an agonist of both CB1R and CB2R, is strongly supported by reports indicating that eCB receptor agonists or inhibitors have an impact on EAE (Baker et al., 2001, Cabranes et al., 2005). Moreover, based on the up-regulation of 2AG as a potential neuroprotective mechanism after mechanical CNS injury (Arevalo-Martin et al., 2010, Panikashvili et al., 2005, Shohami and Mechoulam, 2006), the exogenous administration of 2AG would confer a potential benefit in EAE and/or MS.

Based on previous findings (Shohami and Mechoulam, 2006, Witting et al., 2004a, Witting et al., 2006), the present study addressed the question of whether exogenously administered 2AG might have any effect on the clinical outcome in acute and chronic EAE models, the inflammatory or axonopathic processes, and the expression of CB1R and CB2R in the CNS. In addition, we used in vitro lymph node cell viability assays to test the effect of different 2AG doses under myelin-peptide-selective stimulation.

To our knowledge, this is the first time that exogenous 2AG is applied in acute PLP- and chronic MOG-induced EAE mouse models as an experimental therapy, apart from the study of Baker et al. (2001) in Biozzi mice and studies of endogenous eCD alterations (Centonze et al., 2007a, Witting et al., 2004a). Most importantly, the use of EAE models with either mild or severe clinical course highlights the spectrum of 2AG efficacy in autoimmune demyelination in the CNS.

Section snippets

2AG administration ameliorated clinical course of acute PLP-EAE in SJL/6 mice

The clinical course and disease characteristics of acute PLP-EAE are presented in Fig. 1 (panels A, C, and table included in the figure). Mice developed a monophasic disease without mortality. Control group developed the first clinical signs of EAE on days 11–12, reaching a clinical peak on day 16 (2.75 ± 1.47) (Fig. 1A) with high MMS (table included in the figure). 2AG-treated animals developed a milder disease (Fig. 1A), with significantly delayed disease onset until days 15–16 (Fig. 1C and

Discussion

In the present study, we demonstrated for the first time that exogenous administration of the endocannabinoid 2AG in EAE a) significantly ameliorated both acute and chronic EAE, indicating an impact on acute relapse severity and chronic progressive disability; b) significantly reduced mortality rate in severe MOG-EAE; c) had a significant antiproliferative effect on activated LNCs in vitro, concomitant with increased apoptosis of perivascular infiltrates in vivo; d) ameliorated the

Animal handling

Female SJL/6 and C57/Bl, 8 week-old mice, were purchased from Hellenic Pasteur Institute (Athens) and housed in the pathogen-free animal facility of the B' Neurological Department, AHEPA University Hospital. Animal experimentation was approved by Veterinary Directorate of Thessaloniki and was conducted under compliance with National Institutes of Health guidelines and European Community Directive 86/609/EEC, the Greek Government guidelines, and the local ethics committee. All possible adequate

Acknowledgments

This project has been funded by the General Secretariat for Research and Technology, Greek Ministry of Development, under a “PENED” research grant. Research in Jerusalem was supported by the US National Institute of Drug Abuse (DA-9789 to R.M.). The authors would like to thank Evangelia Nousiopoulou for excellent technical support.

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