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

www.elsevier.com/locate/ynbdi Neurobiology of Disease 30 (2008) 162 – 173

Pattern of axonal injury in murine myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis: Implications for multiple sclerosis Eva Herrero-Herranz,a,1 Luis A. Pardo,a Ralf Gold,b and Ralf A. Linker b,⁎ a

Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Street 3, 37075 Göttingen, Germany Department of Neurology, St. Josef Hospital, Ruhr-University Bochum, Gudrunstr. 56, 44791 Bochum, Germany

b

Received 11 August 2007; revised 17 November 2007; accepted 3 January 2008 Available online 26 January 2008

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

Abbreviations: APP, amyloid precursor protein; Caspr, contactin associated protein; CNS, central nervous system; CNPase, 2',3'-cyclic nucleotide 3'-phosphodiesterase; CNTF, ciliary neurotrophic factor; EAE, experimental autoimmune encephalomyelitis; L, lesion; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NAWM, normal appearing white matter; PFA, paraformaldehyde; p.i., post immunization; PL, perilesion; PLP, proteolipoprotein. ⁎ Corresponding author. E-mail address: [email protected] (R.A. Linker). 1 Present address: Pharmazentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basle, Switzerland. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2008.01.001

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. © 2008 Elsevier Inc. All rights reserved. Keywords: Axonal injury; Neurofilament; Amyloid precursor protein; Sodium channel; EAE; Neurodegeneration

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

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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, 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.

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 Mycobacterium tuberculosis H37RA (Difco, Detroit MI, USA) at a final concentration of 1 mg/ml. Two injections of pertussis toxin (List Biochemicals, Campbell, CA, USA, 400 ng per mouse i.p.) were given at the time of immunization and 48 h later. Animals were weighed and scored for clinical signs of disease on a daily basis using a clinical score as described previously (Linker et al., 2002). Tissue processing At four different time points comprising the beginning (day 9 p.i.), the first maximum (day 13 p.i.) and later phases (day 26 and 40 p.i.) of typical MOG-EAE, groups of three representative mice were taken out of the experiment for histopathological analysis on longitudinal and cross sectioned cryosections. We chose to analyze lumbar spinal cord since this region anatomically best reflects the observed clinical symptoms (paraparesis to paraplegia). Mice were anesthetized with Ketanest (Inresa, Freiburg, Germany) and Rompun (Bayer, Leverkusen, Germany) and transcardially perfused with saline followed by a solution of 4% paraformaldehyde (PFA). Spinal cords were carefully removed, postfixed in the same fixative and then incubated overnight in a solution of 15% sucrose in PBS. Tissue was snapfrozen in methylbutane (−70 °C) and embedded in Tissue-Tek (Sakura, Heppenheim, Germany). 14 μm sagittal or transversal serial sections were collected on gelatin-coated slides by using a Reichert cryotome (Leica, Bensheim, Germany). Histochemistry and immunohistochemistry Formal histopathological evaluation included myelin staining with Luxol Fast Blue and labeling of axons by Bielschowsky silver impregnation. Immune cells were identified by a rat anti-CD4 antibody for CD4 positive T cells, (dilution 1:50, kind gift from Prof. Zinkernagel), a rat anti-CD8 antibody for CD8 positive T cells (1:100, BD, Heidelberg, Germany) and a rat anti-F4/80 antibody for macrophages and microglia (dilution 1:500, Sertoec, Heidelberg, Germany). After inhibition of unspecific binding with 10% BSA, sections were incubated overnight at 4°C with the appropriate primary antibody in 1% BSA. A rabbit anti-rat antibody (1:100, Linaris, Wertheim, Germany) was employed as secondary antibody.

Table 1 Clinical course and time course of inflammation in murine MOG-EAE

Materials and methods 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

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Mean clinical score CD4 positive cells CD8 positive cells F4/80 positive cells

Day 9 p.i.

Day 13 p.i.

Day 26 p.i.

Day 40 p.i.

1.1 ± 0.4 137 ± 32 100 ± 34 640 ± 113

5.4 ± 0.5 478 ± 48 159 ± 45 1257 ± 207

5.1 ± 0.2 217 ± 40** 59 ± 18 472 ± 38**

4.9 ± 0.2 115 ± 39** 95 ± 26 285 ± 42***

Clinical scores and cell numbers are shown as mean ± SEM. ** p b 0.01, *** p b 0.001 in comparison to the respective value on day 13 p.i.

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After blocking of endogenous peroxidase with H2O2, the peroxidase-based ABC detection system (DAKO, Hamburg, Germany) was employed with DAB as the chromogenic substrate. After dehydration, sections were mounted with Entellan for light microscopy.

For fluorescence labeling, sections were blocked with 0.1% Tween-20 and 1% BSA in PBS (Sigma). Primary antibodies were as follows: mouse anti-SMI31 and anti-SMI32 antibodies (dilution 1:1000, Covance, Berkeley, USA), mouse anti-APP (dilution 1:100,

Fig. 1. Axonal loss over the course of murine MOG-EAE. Representative images of transversal lumbar spinal cord sections are shown. Axons are stained by Bielschowsky silver impregnation. Scale bars A and B: 20 μm. (A) At the onset of MOG-EAE (day 9 p.i.) there is already a massive decrease in axonal densities in the lesion (L) as compared to normal appearing white matter (NAWM). (B) At the maximum of disease (day 13 p.i.) and at later disease stages (day 40 p.i.), there is a significant reduction of axonal densities also in the perilesional area (PL). In the late phase of disease (day 40 p.i.) a reduction of axonal densities is even found in the NAWM. (C) Quantification of axon numbers in lesions, perilesional areas, NAWM and grey matter over the course of MOG-EAE. Data are shown as relative axonal densities + SEM. In comparison to the respective numbers in the healthy controls (ctrl.), axonal densities in lesions are reduced at all time points ( p = 0.001 for all time points, see asterisk in figure). In comparison to day 9 p.i., axonal densities in the perilesion and in the NAWM are significantly reduced on day 40 p.i. ( p = 0.0009, see asterisks).

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Fig. 2. Immunoreactivity for APP peaks early during murine MOG-EAE. (A) APP staining (red) is mainly detected in lesions (L) at early time points of the disease (day 13 p.i.) and decreases at later disease stages (day 40 p.i.). Representative images are shown, nuclei are stained in blue. Bar: 45 μm. (B) Quantification of APP positive axons over the course of murine MOG-EAE. Data are displayed as percentage of APP positive axons in relation to silver stained axonal profiles of the respective regions. In comparison to the NAWM, APP immunoreactivity in lesions is significantly increased on day 9 p.i. and day 40 p.i. ( p = 0.004, marked by an asterisk). Note that there is no change of APP positive axons over time in the perilesion and NAWM.

Chemicon, Hofheim, Germany), anti-1.2 and anti-1.6 (dilution 1:50, Chemicon), rabbit anti-Caspr (dilution 1:100, Santa Cruz, Heidelberg, Germany), mouse anti-2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, 1:1000; Sigma, Munich, Germany), rat anti-myelin basic protein (MBP, dilution 1:300, Chemicon), and mouse anti-glial fibrillary acidic protein (GFAP, Cy3 labeled, dilution 1:2000, Sigma). Primary antibodies were incubated overnight at 4 °C. After washing, sections were incubated with the appropriate fluorescent-labeled secondary antibody (Invitrogen, Karlsruhe, Germany or Jackson Laboratories, Suffolk, UK) at a dilution of 1:2000. Specificity of staining was confirmed by omitting the primary antibody. Sections were mounted with ProLong containing DAPI (Invitrogen).

Microscopy and data analysis Sections were analyzed using light microscopy or confocal laser scanning microscopy (Leica, Bensheim, Germany) using the appropriate laser and AOBS configurations for Cy3, Alexa488, DAPI and

Cy5. Areas with complete demyelination and homogeneous immune cell infiltration on defined areas of the anterolateral columns of lumbar spinal cord were identified as lesion, the adjacent tissue with sparse demyelination and few, scattered immune cells as perilesional area and areas of the adjacent tracts without the presence of immune cells or demyelination as normal appearing white matter (NAWM). Axonal densities in grey matter were quantified on defined areas from the lateral anterior horn and medial dorsal horn in the lumbar spinal cord. Blinded quantification of axons and immune cells was performed on serial sections and included each an average of nine spinal cord cross sections from at least three different mice per experimental group. Immune cells were counted by means of overlaying a stereological grid onto the sections and counting inflammatory infiltrates per mm2 white matter. Quantification of demyelinated areas on Luxol Fast Blue stainings was performed semiquantitatively according to Storch et al. (1998). For quantification of axons on spinal cord cross sections, digital images from defined regions of the anterolateral columns were taken with a 63× objective. Subsequently, axons were counted manually from digital images using a grid of 100μm diameter (modified

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after Bitsch et al. (2000); Mews et al. (1998)). Data are presented as percentage of labeled profiles in relation to the total number of silverimpregnated axons in the corresponding lesions. Statistics were performed after checking for normal distribution by Mann–Whitney U test (PRISM software, Graph Pad, San Diego, CA, USA). Differences were considered significant at p b 0.05. Significance is indicated in the figures by asterisks * p b 0.05, ** p b 0.01, *** p b 0.001. Colocalization analyses were performed on digital confocal images. Counting of APP and Nav1.6 or SMI32 double positive axons was done using the Image J plug-in cell counter (http://rsb.info.nih.gov/ij/). The total number of axons positive for APP as well as double positive axons for sodium channels and APP was quantified on lesional and perilesional areas from at least six longitudinal spinal cord sections each representing 0.056 mm2. Results 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 of disease (day 13 p.i.) with a decrease to about 20% of cells at the later disease phase (Table 1). Numbers of macrophages and microglia were about 3–4 fold higher than CD4 positive T cells. In contrast, staining for CD8 positive T cells revealed low cell numbers without significant modulation over the entire disease course (Table 1).

Extensive axonal loss in EAE lesions, but also in the perilesional area, normal appearing white matter and grey matter Actively demyelinating lesions in a defined area of the anterolateral columns of lumbar spinal cord were characterized by the presence of inflammation and demyelination, and axons were counted in lesions, centripetally located perilesional areas as well as NAWM. Axonal densities were then compared to numbers in tracts of healthy mice corresponding to the location of lesions or NAWM. At the beginning of disease, numbers of axons in lesions were already decreased to about 30% of those present in control mice (Figs. 1A and C). With only slight variations, numbers of axons in lesions remained constantly decreased over the course of disease. In contrast, the numbers of axons in the perilesion and also in normal appearing white matter significantly decreased at later time points (day 26 and day 40 pi.) to about 50–60% compared to the respective numbers at the onset of EAE (Figs. 1B and C). Quantification of axonal densities in lumbar spinal cord grey matter

revealed an early decrease on day 9 p.i. of about 25%. At the later disease stages, axonal densities in grey matter remained reduced with a small but not significant increase in comparison to day 9 p.i. In summary, the quantification of silver stained axons speaks for a significant axonal loss in EAE lesions and grey matter early and in the perilesion and normal appearing white matter later in the course of the disease. Immunoreactivity for APP peaks early during MOG-EAE APP has been described as a marker for acute axonal injury in MS and EAE lesions persisting for four weeks (Kuhlmann et al., 2002; Linker et al., 2005). We therefore investigated immunoreactivity for APP over the course of murine MOG-EAE. Early during disease (day 9 and day 13 p.i.) many axons were labeled positive for APP (Fig. 2A). Yet, at later disease stages, numbers of APP positive axons in lesions significantly decreased (Figs. 2A and B). In the perilesion and NAWM, only a fraction of axons displayed APP immunoreactivity without change over time. In summary, relative and absolute numbers of APP positive axons in lesions are high early in MOG-EAE, but decrease after four weeks. Increase of non-phosphorylated neurofilaments over the course of MOG-EAE To investigate the extent of neurofilament phosphorylation over the course of disease, sections were stained with the antibodies SMI31 for phosphorylated and SMI32 for non-phosphorylated neurofilaments (Dahl et al., 1989). In the early phase of MOG-EAE, most axons in the perilesional area and the NAWM were positive for SMI31 (about 40%) and only few axons were immunoreactive for SMI32 (10%). Yet, in lesions, the percentage of SMI32 positive axons was increased while the percentage of SMI31 positive profiles was decreased (Figs. 3B and C). At the later disease phases, there was a large increase in SMI32 positive axons (Figs. 3A and C) while only few axons were labeled positive for SMI31 (Figs. 3A and B). This pattern was also observed in the perilesional area and in the NAWM. In summary, the relative amount of phosphorylated neurofilaments decreases, whilst the percentage of non-phosphorylated neurofilaments increases over the course of MOG-EAE.

Expression of sodium channels Nav1.2 and Nav1.6 in MOG-EAE Studies in animal models as well as MS lesions revealed a redistribution and re-expression of sodium channels in autoimmune demyelination (Coman et al., 2006; Craner et al., 2003b). We investigated the expression of Nav1.2 and Nav1.6 in the spinal cord of murine MOG-EAE. In healthy C57BL/6 mice, Nav1.6 staining is only seen punctually at the node of Ranvier while there is no ex-

Fig. 3. Phosphorylation of neurofilaments over the course of MOG-EAE. (A) Representative images of perilesional areas (PL) at the early (day 9 p.i.) and late stages (day 40 p.i.) of MOG-EAE. Staining was performed with SMI31 for phosphorylated and SMI32 for non-phospohorylated neurofilaments. Over the course of disease, number of SMI31 positive axons decreased while SMI32 positive profiles (red) increased. Bar 20 μm. (B) Quantification of SMI31 positive axons over the course of murine MOG-EAE. Data are presented as percentage of SMI31 positive axons in relation to silver stained axonal profiles of the respective regions. Over time, there is a significant decrease of SMI31 positive axons in the NAWM (day 40 vs. day 9 p.i., p = 0.0002, marked by an asterisk). (C) Quantification of SMI32 positive axons over the course of murine MOG-EAE. Data are presented as in panel B. Over time, there is a significant increase of SMI32 positive axons in the perilesion (day 40 vs. day 9 p.i., p b 0.01) and the NAWM (day 40 vs. day 9 p.i., p = 0.01, see asterisks in figure).

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pression of the embryonic sodium channel Nav1.2 (data not shown). Over the course of murine MOG-EAE, there was a steady increase in Nav1.2 (at day 40 p.i., 82% of all axons in the perilesional area were Nav1.2 positive) and Nav1.6 positive profiles which was more pronounced for Nav1.6 immunoreactivity. Increasing immunoreactivity for sodium channels was not only found in lesions, but in particular in the perilesion and normal appearing white matter at later time points of MOG-EAE (Figs. 4A–C). Nav1.2 and Nav1.6 immunoreactivity was not only punctually located, but distributed longitudinally in transverse and longitudinal sections (Fig. 4A). In summary, Nav1.2 and Nav1.6 sodium channels are increasingly (re)-expressed and redistributed during murine MOG-EAE. Colocalization of sodium channels with APP, non-phosphorylated neurofilaments and Caspr To further characterize the functional relevance of sodium channel expression during murine MOG-EAE, colocalization experiments with different markers for axonal damage and nodal proteins were performed. First, double staining of Nav1.6 with APP as marker of acute axonal damage was performed (Fig. 5A). On blinded quantification at the maximum of disease, 56 ± 11.8% of all APP positive axons were also labeled for Nav1.6 while only 28.6 ± 6.5% of all APP axons were Nav1.2 positive. Thus, more Nav1.6 than Nav1.2 immunoreactive axons also express APP which is well in accordance with earlier observations in a different model (Craner et al., 2004). Next, double labeling for Nav1.6 and SMI32 as marker for non-phosphorylated neurofilaments was performed. At days 9 and 26 p.i., 30% of SMI32-immunoreactive axons were also stained positive for Nav1.6 (Fig. 5B). In contrast, there was only a small overlap of sodium channel expression with the phosphorylated neurofilament marker SMI31 (data shown as supplementary figure). Finally, there was an almost complete overlap in immunoreactivity for Nav1.2 or Nav1.6 with Caspr as another protein of the nodal structure (Fig. 5C). In summary the axonal expression of sodium channels is associated with expression of other nodal proteins and in part also markers for axonal damage. Relation of axonal injury markers to demyelination We were interested in the correlation of markers of axonal injury with the extent of demyelination over the course of murine MOGEAE. To characterize the amount of myelin damage, a Luxol Fast Blue staining was performed. Blinded quantification revealed an increase in demyelinated areas over the course of disease speaking for an incomplete lesion repair and accumulation of myelin damage over time (Fig. 6A). To investigate the relation of sodium channel expression to myelination of axons, double-labeling experiments for CNPase and sodium channels were performed. After obtaining results with Nav1.2 previously (Herrero-Herranz et al., 2007), we

here focused on the sodium channel Nav1.6 (Fig. 6B). Immunoreactivity for Nav1.6 was exclusively present along CNPase negative axons, while axons with intact, CNPase positive myelin did not show re-distribution of sodium channels. As shown in previous studies (Herrero-Herranz et al., 2007), remyelination of MOG-EAE lesions was largely incomplete. Recently, expression of sodium channels in chronic MS lesions was also shown in astrocytes (Black et al., 2007). To investigate the extent of glial expressed Nav1.6 in active MOG-EAE lesion, double labeling of GFAP as an astrocyte marker and Nav1.6 was performed (Fig. 6C). GFAP positive astrocytes displayed only scarce immunoreactivity for Nav1.6 and were mainly Nav1.6 negative. Together with the co-expression of Nav1.6 with Caspr (Fig. 5), these findings speak for a predominant axonal expression of this sodium channel in actively demyelinated lesions. Finally, we studied the relation of non-phosphorylated neurofilaments to the presence of intact myelin. To that end, we performed double-labeling experiments with SMI32 and MBP. Axons positive for SMI32 displayed only thin myelin sheaths or were completely demyelinated. In contrast, axons negative for SMI32 were characterized by an intact myelin sheath. Well in accordance with previous data from MS lesions (Trapp et al., 1998), these results argue for an altered phosphorylation pattern of axons in the presence of demyelination. 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 (Kuhlmann et al., 2002). One may argue that axonal loss at early time points might be simulated by edema occurring with acute inflammation, yet this was corrected for with an appropriate grid for quantification (Bitsch et al., 2000; Mews et al., 1998). The slight increase of axons in lesions on day 13 p.i. and after day 9 p.i. in the grey matter might represent attempted sprouting. Yet, at later time points we could not observe a further increase in axonal densities which may indicate focal regeneration. In summary, the massive loss of axons already occurring at the onset of disease associated with extensive inflammation underscores the relevance of early axonoprotective approaches for treatment of multiple sclerosis (Smith, 2006). Second, our data reveal that axonal loss in the perilesional area and NAWM is extensive at later stages of MOG-EAE and even occurs in grey matter. At later time points, axonal loss in perilesions and NAWM progressed although inflammatory infiltration already significantly declined. In particular, numbers of macrophages, likely

Fig. 4. Expression of axonal sodium channels Nav1.2 and Nav1.6 in murine MOG-EAE. (A) Expression of Nav1.2 in the perilesional area (PL) and NAWM at the maximum of MOG-EAE (day 13 p.i.) and at the late phase of disease (day 40 p.i.). In the perilesional area, Nav1.2 immunoreactivity is already found at the maximum of disease and increases at day 40 p.i. In the NAWM, Nav1.2 expression can be observed at the later stages of MOG-EAE (day 40 p.i.). Representative images from lumbar spinal cords cross sections are shown. Bar 20 μm. (B) Relative number of Nav1.2 positive profiles in relation to the silver stained axonal profiles of the different regions. In lesions, Nav1.2 positive profiles are significantly increased compared to NAWM ( p b 0.05 for day 9, 26 and 40 p.i.; p b 0.001 for day 13 p.i., see asterisks in figure). In comparison of day 9 to day 40 p.i., there is a significant increase of Nav1.2 positive profiles in the perilesion ( p = 0.002, see asterisks in figure). (C) Relative number of Nav1.6 positive profiles in relation to the silver stained axonal profiles of the different regions. In lesions, Nav1.6 positive profiles are significantly increased compared to NAWM ( p b 0.01, for day 9, 13 and 26 p.i., p b 0.5 for day 40 p.i., see asterisks in figure). In comparison of day 9 to day 40 p.i., there is a significant increase of Nav1.6 positive profiles in the perilesion ( p = 0.002) as well as NAWM ( p b 0.001, see asterisks in figure).

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Fig. 5. Colocalization of redistributed sodium channels with APP, non-phosphorylated neurofilaments and Caspr. Representative images of transversal lumbar spinal cord sections are shown. Double-labeled structures are marked by asterisks. Bar represents 20 μm for panels A and C and 45 μm for panel B. (A) Highresolution imaging of immunohistochemical stainings for Nav1.2 or Nav1.6 (green) and APP (red). In a subset of profiles, APP colocalizes with Nav1.2 and even more obvious with Nav1.6. (B) Double labeling of SMI32 with Nav1.6 in lesions and perilesions reveals colocalization in a subset of axons (about 30%). Images were taken at the onset of the disease (day 9 p.i.) and at a later disease stage (day 26 p.i.). (C) Most of the profiles that are Nav1.2 or Nav1.6 positive are also immunoreactive for the nodal protein Caspr (day 26 p.i.).

representing a major culprit for axonal injury (Bitsch et al., 2000) decreased to about one fourth at later disease stages. These data are in accordance with MR spectroscopy investigations revealing axonal dysfunction remote from focal cerebral demyelination (De Ste-

fano et al., 1999). In our model, it might well be possible that axonal loss at lumbar levels is governed by distant rostral lesions in descending fibre tracts leading to dying back of axons. Indeed, inflammatory lesions were present both in the cervical and thoracic

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Fig. 6. Relation of axonal injury to demyelination. (A) Blinded quantification of demyelination on Luxol Fast Blue stainings reveals an increase of myelin damage over time (⁎⁎p b 0.01). (B) Double labeling of Nav1.6 (red) and CNPase (green). A representative longitudinal lumbar spinal cord section is shown (day 26 p.i.). Bar represents 20 μm. Immunoreactivity for Nav1.6 is exclusively present along CNPase negative axons (arrowhead), intact axons with CNPase positive myelin do not show re-distribution of Nav1.6 (asterisk). Sites indicative of putative remyelination are sparse (arrow) and not associated with focal Nav expression. The panel represents a projection of 24 optical sections. (C) Double labeling of Nav1.6 (red) and GFAP (green). A representative transversal section is shown (day 26 p.i.). Bar represents 20 μm. GFAP positive astrocytes display only scarce immunoreactivity for Nav1.6 (arrowheads) and are mostly negative for sodium channels. (D) Double labeling for SMI32 (positive axons in red) and intact myelin sheaths (MBP in green). A representative longitudinal section is shown (day 26 p.i.) Axons positive for SMI32 display only a thin myelin sheath or are completely demyelinated (arrowhead). Axons negative for SMI32 are characterized by an intact myelin sheath (asterisks). The panel represents a projection of 7 optical sections. Bar represents 20 μm.

spinal cord of mice and displayed a similar decrease over time as in the lumbar parts (data not shown). The observed dissociation between decreasing inflammation and increasing axonal degeneration and persisting disability is well in line with the concept that in autoimmune demyelination, inflammatory processes may trigger neurodegenerative processes which at some point continue independently from the immune attack (for review see Bjartmar et al. (2003); Bjartmar and Trapp (2001); Trapp et al. (1999)). Well in line with previous results showing extensive acute axonal damage in CD8:MHC-I deficient mice (Linker et al., 2005), the low numbers of CD8 positive T cells over the course of MOG-EAE underscore the notion that these cells are not critical for axonal injury in this model. Of course, it may well be possible that the quality of the immune infiltrate at the later disease stages differs with respect to the onset of disease. Yet histopathology does not allow further functional insights. Most importantly, motor impairment at later disease stages of MOG-EAE in particular correlates with axonal loss in the perilesion and NAWM rather than in lesions themselves. This axon loss distant from lesions limits axonal plasticity (Kerschensteiner et al., 2004) and might be therefore particularly critical for permanent disability.

In a next step, we investigated the phosphorylation pattern of neurofilaments over the course of MOG-EAE. As markers, we employed SMI31 labeling phosphorylated neurofilaments and SMI32 labeling non-phosphorylated neurofilaments. Earlier studies already disclosed an increase of dephosphorylated neurofilaments in the CSF and in lesions of MS patients (Petzold et al., 2005; Trapp et al., 1998). Well in line with these observations, the prevailing immunoreactivity for SMI32 at later disease stages of murine MOG-EAE speaks for a concurrent dephosphorylation of neurofilaments during disease progression. In particular, an increase of non-phosphorylated neurofilaments also occurs distant from demyelinated lesions. This observation argues for dephosphorylation as functional change in the cytoskeleton of remotely injured axons preceding or paralleling axonal transsections. Interestingly, the sum of SMI31 labeled profiles and SMI32 labeled profiles first increases from day 9 to day 13 p.i., but then decreases at the later disease stages (data not shown). This observation may be explained by the fact that at some point, a subset of axons labeled positive for both, SMI31 and SMI32, while at later disease stages neurofilaments degenerate and are neither labeled by SMI31 nor by SMI32. APP is a well characterized marker for acute axonal injury in neurodegenerative diseases, but also MS (Kuhlmann et al., 2002;

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Pierce et al., 1996). It was shown to accumulate in case of a disturbed fast axonal transport thus again indicating functional changes of damaged axons. Yet, at later disease stages of MOG-EAE, APP immunoreactivity decreases despite ongoing demyelination and axonal loss. Our results indicate that this loss of APP immunoreactivity cannot be solely explained by a loss of axons over time. Rather, structural changes in the axonal cytoskeleton along with persistent injury subsequently result in loss of APP immunoreactivity. In summary, APP is a good marker only for acute axonal injury in demyelinated lesions themselves. Finally, our results reveal a re-expression of the developmentally regulated channel Nav1.2 in murine MOG-EAE and a re-distribution of the sodium channels Nav1.2 and Nav1.6. These channels were shown to be present along damaged axons, but also in glial cells (Black et al., 1994, 2007; Craner et al., 2005). The association of Nav1.2 and Nav1.6 with the paranodal protein Caspr in our model speaks for a re-organisation not only of single proteins, but rather complex nodal structures in particular along axons as already observed in the injured peripheral nervous system (Ulzheimer et al., 2004). These observations are well in line with earlier observations on other models of EAE and recently also MS plaques (Coman et al., 2006; Craner et al., 2003b,a, 2004). Expression of sodium channels may play a role for remyelination (Coman et al., 2006) or constitute an attempt to maintain conduction along demyelinated axons. In actively demyelinating MOG-EAE lesions, we could not delineate a predominant association of sodium channel expression with remyelination or glial cells, but rather found an increase of sodium channels at later disease stages well correlating with an increase in demyelinated areas. These data argue for an incomplete repair and accumulation of lesions over time thus characterizing murine MOG-EAE as a chronic progressive disease rather than a disorder with a persisting deficit after a single demyelinating episode. Of note, the increase in Nav1.2 and Nav1.6 expressions parallels the temporal course of murine MOG-EAE. This observation is well in accordance with a previous study describing the temporal expression of Nav1.8 in Purkinje neurons during chronic-relapsing EAE (Craner et al., 2003a). In view of these results and earlier investigations on murine spinal cord and optic nerve (Craner et al., 2003b), it is tempting to hypothesize that sodium channel reexpression predisposes axons to injury. This notion is well in line with previous in vitro studies suggesting a deleterious role of altered sodium channel expression (Stys, 2005). Increased expression of sodium channels along injured axons may lead to a change in intracellular ion homeostasis leading to an intracellular calcium increase, finally activating harmful proteinases and disturbing the delicately balanced membrane potential and axonal energetic metabolism. Moreover, mitochondrial dysfunction (Dutta et al., 2006) and hypoxic injury (Stys et al., 1992) may contribute to axonal injury. Indeed, Nav1.6 is more extensively expressed at later disease stages and is associated to a higher degree with markers of axonal injury. Here we reveal that axonal loss in murine MOG-EAE is 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 persisting clinical impairment. Our data underscore the value of MOG-EAE in C57BL/6 mice as a model closely mimicking neurodegenerative changes during autoimmune demyelination (Gold et al., 2006). They may indicate which therapeutic approach bears the highest level of success during disease course of EAE, and possibly also MS.

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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