Pattern of axonal injury in murine myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis: Implications for multiple sclerosis

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Abstract

Axonal damage is a correlate for increasing disability in multiple sclerosis. Animal models such as experimental autoimmune encephalomyelitis (EAE) may help to develop better therapeutical neuroprotective strategies for the human disease. Here we investigate the pattern of axonal injury in murine myelin oligodendrocyte glycoprotein peptide 35–55 (MOG) induced EAE. Inflammatory infiltration, axonal densities and expression of amyloid precursor protein (APP), neurofilaments (SMI31 and 32) as well as expression of sodium channels were quantified in lesions, the perilesional area and normal appearing white matter (NAWM). Quantification of T cells and macrophages revealed a significant reduction of inflammatory infiltration at later disease stages despite an increase of demyelinated areas and persistent clinical disability. In lesions, axonal density was already significantly reduced early and throughout all investigated disease stages. A significant axonal loss was also seen in the grey matter and at later time points in the perilesion as well as NAWM. Numbers of axons characterized by non-phosphorylated neurofilaments and re-distribution of sodium channels 1.2 and 1.6 increased over the course of MOG-EAE whilst APP positive axons peaked at the maximum of disease. Finally, double-labeling experiments revealed a strong colocalization of sodium channels with APP, neurofilaments and the axonal nodal protein Caspr, but not glial and myelin markers in actively demyelinating lesions.

In summary, progressive axonal loss distant from lesions is mainly associated with changes in neurofilament phosphorylation, re-distribution of sodium channels and demyelination. This axonal loss is dissociated from acute inflammatory infiltration and markedly correlates with clinical impairment. Consequently, therapeutic intervention may be promising at early stages of EAE focusing on inflammation, or later in disease targeting degenerative mechanisms.

Introduction

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Although morphological changes of axons like transsections and formation of spheroids have long been recognized in MS lesions (Kornek and Lassmann, 1999), the importance of axonal damage was only recently revived and refined with the avenue of new imaging techniques like confocal laser scanning microscopy (Trapp et al., 1998) or magnetic resonance spectroscopy (Narayanan et al., 1997). While histopathologic staining for amyloid precursor protein (APP) as a marker revealed that acute axonal injury in MS is already extensive early (Bitsch et al., 2000, Kuhlmann et al., 2002), imaging studies also point at a critical role of axonal loss for disease progression and accumulation of disability at later time points of the disease (De Stefano et al., 1998, De Stefano et al., 2001).

In rat models, patterns of axonal damage and in some settings also neuronal injury are characterized in experimental autoimmune encephalomyelitis (EAE) after immunization with myelin oligodendrocyte glycoprotein (MOG) mimicking many aspects of MS (Hobom et al., 2004, Storch et al., 1998). After immunization with recombinant MOG, axonal damage was observed in the white matter of marmoset monkeys (Boretius et al., 2006) and rodents (Kornek et al., 2000) and in focal EAE models also in the cortex of susceptible rat strains (Merkler et al., 2006). Investigation of axonal damage in chronic rat EAE and murine relapsing remitting proteolipoprotein (PLP) induced EAE underscored the concept that axonal loss may in particular correlate with permanent disability (Papadopoulos et al., 2006, Wujek et al., 2002). In murine MOG-EAE induced by the encephalitogenic peptide 35–55 (MOG 35–55 EAE), axonal injury was mainly investigated in genetic models. Mice with a deficiency of the axonoprotective neurotrophic cytokine ciliary neurotrophic factor (CNTF) exhibit a more severe course of MOG-EAE with enhanced myelin and axonal pathology (Linker et al., 2002). In a Thy1-GFP transgenic mouse model, patterns of axonal damage could be directly characterized by fluorescent labeling of axon tracts in the spinal cord (Bannerman et al., 2005).

Yet, EAE models do not only allow investigation of axonal injury, but also recovery and axonoprotection (Diem et al., 2007). In a focal EAE model in rats, remodelling of axonal connections could be elegantly investigated (Kerschensteiner et al., 2004). Recent studies focused on the role of the wlds fusion protein as an endogenous axonal protection mechanism in murine MOG-EAE (Kaneko et al., 2006, Tsunoda et al., 2007) while some earlier studies in rodents characterized neuroprotective treatment approaches like blocking of glutamate receptors (Pitt et al., 2000).

Different mechanisms contribute to axonal damage during autoimmune demyelination (for overview see Neumann (2003)). Among others, candidates include TNF-alpha mediated cytotoxicity, Fas–FasL interaction or glutamate excitotoxicity (Werner et al., 2001). Moreover, interaction of cytotoxic CD8 positive T cells with upregulated MHC-I on axons may play a role (Medana et al., 2001) although mice lacking MHC-I exhibit significant amounts of APP positive axons (Linker et al., 2005). In a murine EAE model, an altered expression of sodium channels was shown in the optic nerve (Craner et al., 2003b). At sites of axonal injury, this re-expression and re-distribution of sodium channels correlated with APP expression as a marker of axonal damage and expression of a sodium–calcium exchanger (Craner et al., 2004). Indeed, blocking of sodium channels may provide an attractive new therapeutic approach for autoimmune demyelination (Bechtold et al., 2004, Bechtold et al., 2006, Black et al., 2006). Recently, the expression of sodium channels in MS and EAE lesions was also implicated in remyelination (Coman et al., 2006). Yet, altered expression of sodium channels may also be detrimental: Changes in intracellular sodium could lead to reverse action of a sodium–calcium exchanger thus resulting in calcium overload and subsequent destruction of the axonal cytoskeleton (for review see Stys (2005)). The importance of calcium for axonal degeneration is further underscored by a study revealing expression of calcium channel subunits in dystrophic axons (Kornek et al., 2001).

Despite all these investigations, a comprehensive analysis including different markers of axonal damage in MOG-EAE of C57BL/6 mice has not been undertaken so far, although the model is of particular interest in view of the many genetically engineered strains on this background. Here we investigate axonal injury over the course of MOG-EAE in C57BL/6 mice including quantification of axonal loss, APP expression, neurofilament phosphorylation and distribution of sodium channels. Our data reveal distinct patterns of axonal pathology early and late during MOG-EAE.

Section snippets

Animals

C57BL/6 mice were purchased from Harlan (Borchen, Germany) and bred at the in-house animal care facilities of the Institute for MS Research, University of Göttingen, Germany. All animal experiments were performed in accordance with the Lower Saxony State regulations for animal welfare.

Induction and clinical evaluation of active MOG-EAE

For active induction of EAE, a total of 22 mice received a s.c. injection at flanks and tail base of 200 µg MOG 35–55 peptide (Charité, Berlin, Germany) in PBS emulsified in an equal volume of CFA containing

Clinical course and pattern of inflammation in murine MOG-EAE

After onset of disease shortly before day 10 p.i. and the first maximum around day 13 p.i., mice display persistent disability over time remaining unchanged upon observation until day 60 p.i. (Table 1). To investigate inflammation, sections were stained with the anti F4/80 antibody to label macrophages and microglia and an anti-CD4 or anti-CD8 antibody to label CD4 positive or CD8 positive T cells. Both numbers of macrophages and microglia as well as CD4 positive T cells peaked at the maximum

Discussion

In the present study we investigate time course and pattern of axonal injury in murine MOG-EAE in C57BL/6 mice. First, we show that axonal loss in lesions, but also in grey matter occurs very early and does not recover over time. Well in line with the observation of early axonal injury in animal models (Wang et al., 2005) human studies investigating the extent of acute axonal damage in MS lesions by APP immunohistochemistry also revealed extensive damage already at early disease stages (

Acknowledgments

This work was supported by the Gemeinnützige Hertie-Stiftung (project 1.01.1/05/009), the Max-Planck-Society and the Deutsche Forschungsgemeinschaft (through SFB 406). The skillful technical assistance of Silvia Seubert and Alexandra Bohl is gratefully acknowledged. We thank Profs. W. Stühmer and C. Stadelmann for the advice and stimulating discussions.

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    Present address: Pharmazentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basle, Switzerland.

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