Animals and in vivo injections. All the studies comply with the George Washington University Medical Center Institutional Animal Care and Use Committee guidelines. Both male and female MBP-iCP9 transgenic mice [10] on a C57Black6 background were used throughout this study. Due to the limited number of transgenic pups, we were unable to perform gender specific studies although no obvious differences were detected. For the EAE induction studies, only female mice were used per manufacturer’s instruction (Hook Laboratories; # EK-2110).
Pups were injected subcutaneously with 100 mg/Kg body weight of CID (clontech laboratories; #635069) daily for 7 days starting on postnatal day 4 or every other day for a total of 7 injections. CID stock solution was made in 100% ethanol and diluted in equal volume of Polyethylene Glycol (PEG400, Fisher Scientific; #P167) and 1% Tween-20 in PBS. Controls were injected with vehicle lacking CID, where 100% ethanol was added to PEG400 and 1% Tween-20 in PBS. Under terminal sedation, mice were sacrificed 2 weeks after the LPC and 4 weeks after the EAE studies and transcardially perfused with 1xPBS followed by fixative. Animals were fixed with either 4% paraformaldehyde (PFA) for immunefluorescence staining or 4% paraformaldehyde-2% glutaraldehyde-0.1 M sodium Cacodylate (PFA/GA) for Scanning Electron Microscopy (SEM). A minimum of 3 animals were used for each study.
LPC surgeries. Male and female pups were injected with vehicle or CID, allowed to mature to 6-7 weeks of age, and a spinal cord LPC lesion performed at T10 and T11 as previously described [11]. Animals were allowed to recover for 14 days before analysis.
EAE induction. Female vehicle or CID injected pups were allowed to mature to 10-11 weeks at which point EAE was induced accordingly to the manufacturer’s guidelines (Hooke Laboratories #EK-2110). Animals showed symptoms of EAE approximately 8 days after induction and were scored on daily basis according to the following criteria: 1 loss of tail tonicity or hind limb weakness to 5 severe paralysis or death (Table 1). Animals scored 3 or higher were given 0.2 ml saline to prevent dehydration.
Open field testing. For functional testing, mice were placed in a Plexiglas open field (Med Associates, St Albans, VT, USA) outfitted with photobeam detectors, and their activity was monitored using the activity monitoring software (Med Associates). Mice were allowed to habituate in the open field for 10 min and total distance traveled and speed were recorded [14].
Tissue processing. Tissues were processed for 3 different procedures. 1) Immunofluorescence staining: animals were fixed with PFA and spinal cords were cryoprotected using 10, 20, and 30% sucrose gradient in 1xPBS solutions; sections were cut at 20 µm. 2) Electron Microscopy: animals were fixed with PFA/GA and spinal cords were cut to 400 µm sections before processing. Sections were then osmicated (1% OsO4) for at least 4 hours followed by 1% uranyl acetate overnight. After gradated dehydration with ethanol and propylene oxide, sections were placed in Epon812 and cut at 1 µm or 120 nm using an ultramicrotome (Leica UC6). Some sections were stained with Solochrome or Toluidine Blue staining to visualize myelin and some were placed on semiconductor grade Si-wafer microtome. 3) Immunoblotting: animals were perfused with cold 1xPBS and spinal cords were flash-frozen in liquid nitrogen and stored at -80 °C.
Immunostaining. Cross sections were rehydrated, blocked, and incubated with primary antibodies overnight followed by appropriate secondary antibodies for an hour prior to mounting. The following primary antibodies were used: MBP (1:300- Abcam; #7349), DsRed (1:400- Takara; #632496), Iba1 (1:500- WACO; #019-19741), and GFAP (1:500- Biolegend; #PRB-571C), PDGFαR (1:100- BD Biosciences; #558774), and CC1 (1:200- Millipore; #OP80). Appropriate secondary Alexa 488 or 594 (1:500) antibodies were used. Sections were counter-stained with Dapi (1:1000- Thermofisher; #46190) before mounting (Fluoromount G: Electron Microscopy Sciences; #17984‐25).
Immunoblotting. Frozen tissues were processed and lysates were run on 4-20% gels (BioRad; #456-1094). The following primary antibodies were used: MAG (1:5000, Generous gift from Dr. Richard H. Quarles [15]), MBP (1:1000 for WB; Abcam; #7349), and Actin (1:2000; Santa Cruz; #SC47778) antibodies. Appropriate secondary antibodies were used at 1:5000.
Microscopy. Microscopy was performed at the Center for Microscopy and Image Analysis (CMIA) at the George Washington University Medical Center. Confocal microscopy images were captured using the Zeiss Cell Observer Z1 spinning disk confocal microscope (Carl Zeiss, Inc.) equipped with ASIMS-2000 (applied scientific Instrumentation) scanning stage with z-galvo motor, and Yokogawa CSU-X1 spinning disk. Zen Blue software (Carl Zeiss, Inc.) was used with 25x and 63x objectives to acquire tile images and produce maximum intensity projections. SEM was performed using a Helios NanoLab 660 SEM (Thermo Fisher, FEI) equipped with a concentric backscattering detector (CBS) using immersion mode for SEM high-resolution imaging. For the acquisition conditions, we used 2 kV with a landing current ranging from 0.2-0.4 nA with a working distance of 4 mm. For imaging, the entire spinal cord was tile-imaged at low magnification (600x) and fused into a single tiling map per sample and was used as a navigation map to identify lesioned areas (MAPS software). Then, high-resolution (3,500x or 15,000x) imaging was performed in the focus area at 10 ms dwell time and 20 mm horizontal field of view.
Statistical Analyses. All statistical tests were performed using the GraphPad Prism Program, Version 6 (GraphPad Software, Inc. San Diego, CA). A p value<0.05 was considered statistically significant and is demarked with an asterisk. NIH Image J was used to analyze pixel intensity for Western blot analysis. Photoshop was used to calculate the percent lesion and to count cells and the number of remyelinated axons in 3 random sites of the spinal cord under 63x magnification.