Mice, Virus and Ribavirin Inoculations
SOD1G85R (B6.Cg-Tg (SOD1G85R)148Dwc/J; JAX stock #008248) and its background strain C57BL/6J (JAX stock #000664) mice were obtained from Jackson Laboratory (Maine, United States). The neonates (2–3 days old) of SOD1G85R or C57BL/6J mice were intracerebroventricularly inoculated with either a non-lethal dose (5×102 pfu (plaque-forming units) in 2 µl volume) of CVB3 (Kandolf strain) or an equal volume of Dulbecco's modified Eagle’s medium (DMEM, mock infection). All animal procedures were approved by the University of British Columbia Research Ethics Board (Animal Care #A20-0156 and A17-0227).
Ribavirin (Sigma-Aldrich, St. Louis, MO; R9644) treatment in CVB3-infected SOD1G85R mice was performed by subcutaneously injecting either ribavirin (100 mg/kg mouse body weight) diluted in DMEM or an equal volume of DMEM in the neck of neonatal mice. The injections were done every 3 days starting at either day 1 or day 15 post-infection (PI) for a total of 5 injections.
Mouse Motor Function Tests
After 20-week mock or CVB3 inoculation, SOD1G85R (B6.Cg-Tg(SOD1G85R)1248Dwc/J) and C57BL/6J mice underwent weekly motor function tests (hindlimb reflex, inverted grid test, and gait analysis) until either full disease paralysis or 60 weeks post-injection as described [28, 29].
Hindlimb reflex was performed by lifting the mice from the tip of the tail to assess and score their hind leg reflex on a scale of 4–0 (4 = healthy animal with full extension of both hind legs, and 0 = both limbs are fully clasped). Reduction in leg extension is an early deficit observed in mutant SOD1 transgenic mice.
An inverted grid test was performed by placing the mice on an inverted metal grid (10 cm above the surface) and measuring the time (in seconds, with the maximum monitoring time as 120 seconds) for the mice to fall. The test is used to assess the arm/leg strength of the mice .
Gait analysis is a test that quantifies the mouse movement based on its gait distance. In the assessment, the mouse feet were first marked by non-toxic paints and then placed on a long paper (60 cm long, 20 cm wide) that was covered by cardboard, allowing the mice to walk forward in one direction. After the paints were dried, the stride distance (the distance of forward movement between each stride) was measured and recorded. In addition to the gait measurements, the time in seconds (footprint time) needed for each mouse to walk over the track was recorded as an additional measurement of the animal’s motor function .
Tissue Preparation And Immunohistochemistry Staining
Mouse brains harvested were fixed with 4% formaldehyde and paraffinized. Paraffin-embedded sections (4 µm thickness) were deparaffinized through xylene and a series of isopropanol solutions (100%, 90%, and 70%). Hematoxylin and eosin (H&E) staining was conducted to evaluate tissue damages, while immunohistochemical (IHC) staining was used for detecting the presence and localization of proteins of interest. For IHC, antigen retrieval was done by heating tissue sections in pH 6.0 citrate buffer (Life Technologies Carlsbad, CA; 005000) for 25 minutes at 121oC, and then peroxidase blocked using 30 mg/ml hydrogen peroxide solution. Multiple washes with 1⋅ TBS pH 7.6 (Tris-buffered Saline, 0.05M Tris, 0.155M NaCl) were performed afterward. The MACH4 Universal HRP-Polymer Detection System (Biocare Medical, Pacheco CA; BRI4012H) was used according to the manufacture’s procedure for IHC staining experimentations. After the TBS washes, tissue sections were incubated with primary antibodies (dilution and targets are shown below) overnight at 4oC, and counterstained at the end with hematoxylin solution Gill II (Sigma-Aldrich, St. Louis, MO; GHS232).
Mouse brain tissues were immunostained using the follow primary antibodies diluted in TBS-PBS buffer (1% BSA, 1.5M NaCl, 0.5M pH 7.6): VP1 (1:2000 dilution, IgG2a monoclonal antibody, Mediagnost, Reutlingen, Germany; M47), GFAP (1:200 dilution, mouse monoclonal antibody, StressMarq, Victoria, BC, Canada; SMC-441), Iba1 (1:200 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-32725), TDP-43 (1:1000 dilution, rabbit polyclonal antibody, Proteintech, Rosemont, IL, USA; 10782-2-AP), SQSTM1/p62 (1:200 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-28359), ubiquitin (1:2000 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-8017), CD68 (1:200 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc20060), CD19 (1:500 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-373897), CD79A (1:300 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-20064), CD4 (1:500 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-19641), CD8 (1:500 dilution, mouse monoclonal antibody, Santa Cruz Biotechnology, Dallas, TX, USA; sc-1177), and NK1.1 (1:1000 dilution, mouse monoclonal antibody, eBioscience, San Diego, CA, USA, 14-5941-82). Images were taken using the Aperio ScanScope AT (Digital slide scanner, Leica Biosystems Inc., Buffalo Grove, IL, USA).
Rna Extraction And Gene Expression Assays
Mouse brain tissues were homogenized using Qiagen Tissuelyzer LT, and RNAs were extracted using the Monarch total RNA miniprep kit (New England Biolabs, T2010) following the manufacturer’s instructions. Gene expression was measured using NanoString Mouse Immunology Panel (561 targets with 15 internal reference targets) that was ran on a NanoString nCounter® Profiler (NanoString Technologies Inc., Seattle, WA, USA) according to the manufacturer’s instructions. The mRNA levels of different proinflammatory genes were measured via RT-qPCR using the Luna universal one-step RT-qPCR kit (New England Biolabs, E3005) on a QuantStudio 6 Pro real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primer pairs used for mRNA measurement in mouse tissues are as follows: TNFA (forward, 5’- GTC CCC AAA GGG ATG AGA AGT T -3’; reverse, 5’- GTT TGC TAC GAC GTG GGC TAC A -3’), NFKB2 (forward, 5’- TGC TGA TGG CAC AGG ACG AGA A -3’; reverse, 5’- GTT GAT GAC GCC GAG GTA CTG A -3’), CXCL10 (forward, 5’- GCT GGG ATT CAC CTC AAG AA -3’; reverse, 5’- CTT GGG GAC ACC TTT TAG CA -3’), CCL2 (forward, 5’- GCT ACA AGA GGA TCA CCA GCA G -3’; reverse, 5’- GTC TGG ACC CAT TCC TTC TTG G -3’), CCL5 (forward, 5’- GCT GCT TTG CCT ACC TCT CC -3’; reverse, 5’- TCG AGT GAC AAA CAC GAC TGC − 3’), IL6 (forward, 5’- ACA ACC ACG GCC TTC CCT AC -3’; reverse, 5’- TCT CAT TTC CAC GAT TTC CCA G -3’), IL10 (forward, 5’- CCC ATT CCT CGT CAC GAT CTC − 3’; reverse, 5’- TCA GAC TGG TTT GGG ATA GGT TT -3’), β-actin (forward, 5’- CAT TGC TGA CAG GAT GCA GAA GG -3’; reverse, 5’- TGC TGG AAG GTG GAC AGT GAG G -3’).
Quantification And Statistical Analysis
Quantification of immunohistochemistry images was performed using ImageJ (version 1.0) with the combination of Colour Deconvolution Plugin (version 1.5) (http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html - accessed in November 2017) to generate the optical density value based on the intensity of the staining as described . Statistical analysis and the corresponding graphs were done using GraphPad Prism 8 V8.4. Further details in statistical analysis are described in Figure Legends.