Materials & Methods

Animals and virus infection

Five-week-old female SJL/JHanHsd-mice (Harlan Winkelmann, Borchen, Germany) were housed as described [1]. Intracerebral infection was carried out under general anesthesia with medetomidin (0.5mg/kg; Domitor®; Pfizer GmbH, Karlsruhe, Germany) and ketamine (100mg/kg; Ketamin 10%, WDT, Garbsen, Germany). Euthanasia was performed with a 5-fold overdose of the same anesthetics. Mice were inoculated into the right cerebral hemisphere with 1.63x106 PFU/mouse of the BeAn-strain of TMEV in 20µl Dulbecco`s Modified Eagle Medium (PAA Laboratories, Cölbe, Germany) with 2% fetal calf serum and 50µg/kg gentamycin. Mock infected mice received 20µl of the dilutent only. Groups of five to six TMEV-infected and mock-infected mice were killed 1 hour (h) and 7, 14, 28, 42, 56, 70, 98, 147, and 196 days (d) pi. The spinal cord was immediately dissected, the cervical segments 1-2, the thoracic segments 3-4 and the lumbar segments 1-2 were fixed in 10%

formalin for 24 h, decalcified in 25% dinatrium-ethylenediaminetetraacetic solution for 48 h and embedded in paraffin wax. The remaining parts of the spinal cords were removed from the spinal canal and immediately snap frozen and stored at -80°C as described [37]. At 14, 42, 98, 147 and 196 dpi additional groups of six TMEV-infected and mock-infected mice were killed and the spinal cords were immersion fixed in 5% glutaraldehyde / cacodylate buffer.

All animal experiments were authorized by the ethical commission for animal rights and the local authorities (Regierungspräsidium Hannover, Germany, permission numbers: 509c-42502-02/589 and 33-42502-05/963).

Footprint analysis

The clinical course of TME was evaluated with footprint analysis, a special test to evaluate motor strength and coordination [38, 39]. Groups of 10 - 12 mock-infected or TMEV-infected mice were repeatedly assayed at 0 (pre-infection), 28, 56, 98, 147 and 196 dpi by footprint analysis. The hindlimb paws were painted with blue nontoxic food paint (Schwartauer Werke, Bad Schwartau, Germany), and the mice moved along an inclining walkway (100.0 cm length, 4.5 cm width, 30° inclination) that was lined with a strip of white paper leading to a dark room. After drying, the prints were digitized using a handheld scanner (Logitech ScanMan color, Logitech Europe, Romanel-sur-Morges, Switzerland) and the length of 8-10 strides per animal and day were measured using the computer program FOOTPRINTS [38].

The percental change of stride length compared to the baseline measurement at 0 dpi was calculated for every animal to correct for individual differences.

Semithin sections and electron microscopy

Transversal segments of the thoracic spinal cord were fixed in 5% glutaraldehyde / cacodylate buffer for 24 h, followed by postfixation with 1% osmium tetroxide. Following dehydration in a graded series of alcohol, sections were embedded in epoxy resin as described [40]. 1 μm thick semithin sections were stained with toluidine blue and evaluated with an Axiophot microscope (Zeiss, Oberkochen, Germany) using a 100-fold oil immersion objective. The degree of de- and remyelination was semiquantitatively evaluated as follows: 0 = no changes, 1 = from single foci up to 1/4 of the white matter affected, 2 = from 1/4 up to 1/2 of the white matter affected, 3 = more than 1/2 of the white matter affected. Ultrathin sections were cut from selected representative specimens, stained with uranyl acetate and lead citrate and viewed using a Zeiss EM 10C electron microscope (Zeiss, Oberkochen, Germany).

Immunohistology

Immunohistology was performed as described [1]. Briefly, 4 µm thick transversal sections of the formalin-fixed, paraffin embedded thoracic spinal cord were dewaxed and hydrated through graded alcohols. Endogenous peroxidase activity was quenched by 0.5% H2O2. For demonstration of NG2, CNPase, and nonphosphorylated neurofilament (non-pNF) sections were heated in 10 mM Na-citrate-buffer pH 6.0, for 20 minutes (min) in a microwave oven (800 W). Thereafter, sections were incubated with 20 % goat serum for 30 min to block non-specific binding sites prior to incubation with primary antibodies (NG2, polyclonal rabbit anti-rat, diluted 1:200, Chemicon Europe, Hofheim/Taunus, Germany; GFAP, polyclonal

rabbit anti-cow, diluted 1:2000, Dako Diagnostika, Hamburg, Germany; non-pNF, monoclonal mouse IgG1, clone SMI 311, diluted 1:8000, Sternberger Monoclonal Inc., Baltimore, MD, USA) at 4°C overnight. This was followed by a biotinylated goat anti-rabbit IgG or goat anti-mouse IgG respectively (H+L; Vector Laboratories, Burlingame, CA, USA) and the avidin-biotin-peroxidase (ABC)-complex (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Positive antigen-antibody reactions were visualized by incubation with 0.05% 3,3’-diaminobenzidine-tetrahydrochloride- 0.03% H2O2 in 0.1M imidazole, pH 7.08 for 10 min followed by slight counterstaining with Mayer’s hemalaun. For detecting CNPase (monoclonal mouse anti-human IgG1, clone 11-5B, diluted 1:200, Chemicon Europe, Hofheim/Taunus, Germany) the Dako Ark animal research kit (Dako Diagnostika, Hamburg, Germany) was used according to the manufacturer`s instructions.

Controls were incubated with normal rabbit serum (Sigma-Aldrich Chemie, Taufkirchen, Germany) or a mouse IgG1 anti-isotype antibody (Chemicon Europe, Hofheim/Taunus, Germany) instead of the primary antibodies. The CNPase-negative area was measured after manually outlining the total white matter plane, employing the analySIS®3.1 software package (SOFT Imaging system, Münster, Germany). The percentage of spinal cord demyelination was determined by substracting the mean CNPase-negative white matter in TMEV-infected from mock-infected mice. Similarly, the percentage of lesioned white matter area containing inflammation and demyelination was determined using Luxol fast blue-Cresyl violet (LFB-CV) stained serial sections. The density of NG2, CNPase and GFAP positive cells / mm2 was manually counted in randomly selected thoracic spinal cord white matter areas with a 40-fold objective and a 10-fold calibrated eyepiece reticule (Olympus Europe, Hamburg, Germany). For every spinal cord, a mean area of 0.15 mm2 was counted for normal appearing white matter (NAWM) and lesioned white matter, respectively.

Immunofluorescence and confocal laser scanning microscopy

A sequential immunofluorescence-staining technique was used for double-labeling studies. 8 µm thick methanol-fixed frozen sections of the spinal cord were washed with 0.1% Triton X-100 in PBS, and nonspecific binding was blocked by incubation with 20 % goat serum for 30 min prior to the incubation with the antibody against NG2 (polyclonal rabbit anti-rat, diluted 1:200, Chemicon Europe, Hofheim/Taunus, Germany) at 4°C overnight. After washing, an incubation with a Cy3 conjugated goat anti-rabbit IgG antibody (H+L, diluted 1:200, Jackson Immuno Research Europe, Newmarket, United Kingdom) was performed for 1 h. After additional washing steps, sections were incubated with the second primary antibody (CNPase,

monoclonal mouse anti-human IgG1, diluted 1:3200; Chemicon Europe, Hofheim/Taunus, Germany; GFAP, monoclonal mouse anti-pig IgG1, diluted 1:3200, Sigma-Aldrich Chemie, Taufkirchen, Germany) for 2 h at room temperature. These second primary antibodies were labeled with Alexa Fluor 488 conjugated anti-mouse-IgG1 Fab-fragments at a molar ratio of 1:6 according to the manufacturer’s instructions (Zenon Mouse IgG labeling kit, Invitrogen, Karlsruhe, Germany). After further washing steps, sections were post-fixed in 4%

formaldehyde, counterstained using 1.0% bisbenzimide and mounted with fluorescent mounting medium (Dako Diagnostika, Hamburg, Germany). Controls were incubated with normal rabbit serum (Sigma-Aldrich Chemie, Taufkirchen, Germany) and/or a mouse IgG1 anti-isotype antibody (Chemicon Europe, Hofheim/Taunus, Germany) instead of the primary antibodies. Labeled sections were analyzed using a Fluoview FV-1000 confocal laser scanning biological microscope (Olympus Europe, Hamburg, Germany) equipped with a 60-fold water-immersion objective as described [41]. For the excitation of Alexa Fluor-488 the 488 nm line of a multi-line argon laser, for the excitation of Cy3 a 543 nm helium-neon laser, and for the excitation of bisbenzimide a 405 nm diode pumped solide-state laser was used, respectively. Each laser line was scanned separately for individual excitation of the dyes, and the data sets were finally merged and analyzed employing the FV10-ASW® 1.4a Fluoview software (Olympus Europe, Hamburg, Germany). The images in the figures (Figs.) represent single optical sections of ~0.35 µm thickness.

RT-qPCR

RNA was isolated from 40 mg frozen spinal cord tissue with the Absolutely RNATM RT-PCR Miniprep Kit (Stratagene® Europe, Amsterdam, Netherlands) according to the manufacturer’s recommendations. RNA concentration was measured at 260nm using a spectral photometer (GeneQuant pro, Amersham Biosciences Europe GmbH, Freiburg, Germany) and final RNA concentration was set to 20 ng/μl. RNA was reversely transcribed employing the OmniscriptTM Reverse Transcriptase Kit (Qiagen GmbH, Hilden, Germany), 10 μM random hexamers (Random Primers; Promega GmbH, Mannheim, Germany), and 0.5 U/μl RNAse-inhibitor (RNaseOUTTM, InvitrogenTM GmbH, Karlsruhe, Germany) according to the manufacturer’s protocol. Primers for the detection of NG2, CNPase, total MBP mRNA (MBPtotal; primers located in the exons 3 and 3/4), and exon 2 containing splice variants of MBP mRNA (MBPexon2; primers located in the exons 1 and 2/3) were selected from the murine GenBank™/NCBI reference sequences using the computer program Primer Designer version 2.0 (Scientific & Educational Software, Cary, NC, USA; sequences are

available in Anhang 10.1, supplemental table 4-1). Primers for detecting the mRNA of GFAP [42], PDGFα-R [43], the four housekeeping genes GAPDH, ß-actin, HPRT, SDHA, and TMEV RNA were taken from the literature [37]. Quantitative PCR was performed with 1 μl cDNA per 25 μl reaction employing the Brilliant® SYBR® Green QPCR Master Mix Kit (Stratagene® Europe, Amsterdam, the Netherlands) on a MX3005P® Multiplex Quantitative PCR System (Stratagene® Europe, Amsterdam, the Netherlands) with an absolute external cDNA standard containing 102-108 copies/µl as described [37]. The specificity of positive reactions was confirmed by melting point analysis. The obtained data were analyzed using the MX3005P® software version 2.02 (Stratagene® Europe, Amsterdam, the Netherlands). Data were normalized with a normalization factor achieved by geometric averaging of all four housekeeping genes using the geNorm software version. 3.4 [44].

Statistics

Statistical analysis was performed using SPSS for Windows version 13.0. The normalized copy numbers / 10 ng RNA were log10-transformed prior to statistical analysis to obtain normal distribution. RT-qPCR samples without measurable mRNA (no Ct) were set to detection limit / 2. Statistical differences were evaluated employing a two-way ANOVA for the factors group (TMEV-infected or mock-infected) and time (time pi) with post-hoc independent t-tests for the different timepoints. If appropriate, a two-way repeated measures ANOVA was used. For the categorical data, a Mann-Whitney U test was used. For the correlation between histological and molecular data, the mean cellular density per spinal cord section was calculated for NG2, CNPase and GFAP positive cells. For linear correlation, Pearson’s product moment correlation coefficient was calculated. In general, statistical significance was designated as p ≤ 0.05.