All mice described in this study were adult C57BL/6 males, and were housed in a pathogen-free facility. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of California Davis. RAG1−/−
/J, Jackson Lab #002216) were used to examine innate immune responses in the absence of adaptive immunity; these mice lack the capacity to produce mature T or B lymphocytes (Mombaerts et al, 1992
). Thy1-STOP-EYFP mice (B6.Cg-Tg(Thy1-EYFP)15Jrs/J, Jackson Lab #005630), in which Thy1 promoter-driven expression of enhanced yellow fluorescent protein (EYFP) in neurons was prevented by a loxP-flanked STOP sequence, were bred with Emx1Cre mice (B6.129S2-Emx1tm1(Cre)Krj
/J, Jackson Lab #005628), in which Cre is constitutively expressed in forebrain neurons, to generate offspring in which, in spinal cord, corticospinal axons, but not other spinal cord axons, were selectively labeled with EYFP (Bareyre et al, 2005
MOG peptide-EAE was induced in 3 month postnatal C57BL/6 mice by subcutaneous flank administration of 300 µg of rodent MOG peptide (amino acids 35–55, New England Peptides) in CFA containing 5 mg/ml killed Mycobacterium tuberculosis (Difco) on day 0, with intraperitoneal administration of 75 ng of pertussis toxin on days 0 and 2. “CFA control mice” received CFA and pertussis toxin, but no MOG peptide, and normal control mice received no injections. The mice were weighed and examined daily. Neurological deficits were graded on a 5 point scale (limp tail or waddling gait = 1; limp tail and waddling gait = 2; single limb paresis and ataxia 2.5; double limb paresis = 3; single limb paralysis and paresis of second limb = 3.5; full paralysis of 2 limbs = 4; moribund = 4.5; and death = 5) (Zhang et al, 2003
; Bannerman et al, 2005
Isolation of leukocytes from mouse spleen/lymph nodes and CNS
Mice sacrificed by CO2 asphyxiation were perfused with ice cold PBS. Spleens and draining lymph nodes were harvested, combined, minced in PBS, and pushed through a 40 µm mesh. Red blood cells were lysed with ACK solution (Quality Biologicals). Brains and spinal cords were minced, digested at 37°C for 30 min in PBS containing 0.04 units of Liberase R1 (Roche) and 10 µg of DNase I (Roche) per ml. Softened fragments were pushed through a 100 µm mesh. Mononuclear cells from spleen/lymph nodes and from CNS were isolated via a discontinuous 40/70% (v/v) Percoll gradient.
Ex vivo T cell responses
Mixed splenocytes and lymph node cells were cultured in 200 µL of RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 U penicillin-streptomycin, 50 µM 2-mercaptoethanol, and 1 mM sodium pyruvate with or without 50 µg/ml MOG peptide (amino acids 35–55) for 24 hrs. The cells were incubated with brefeldin A (GolgiPlug, BD Bioscience) or brefeldin A plus ionomycin (Calbiochem, 750 ng/ml) and phorbol 12-myristate 13-acetate (PMA; 50ng/ml, Sigma-Aldrich) for the last 5 hr (Park et al, 2005
Mixed splenocytes and lymph node cells were immunostained after the 24 hour culture described above. CNS mononuclear cells were immunostained after incubation at 37°C for 3 hours in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin-streptomycin, 50 µM 2-mercaptoethanol, and 1 mM sodium pyruvate in the presence of brefeldin A. Immediately prior to immunostaining, Fc receptors were blocked for 10 min with anti-CD16/32. For Th1/Th17 lymphocyte analysis, cells were stained with Pacific Blue-labeled anti-mouse CD4, fixed, permeabilized using a Cytofix/Cytoperm Plus Kit according to the manufacturer’s protocol, and stained with allophycocyanin (APC) labeled anti-mouse IFN-γ and phycoerythrin-labeled anti-mouse IL-17 (all reagents from BD Bioscience). For regulatory T lymphocyte (Treg) analysis, cells were stained with Pacific Blue-labeled anti-mouse CD4 (BD Bioscience) and APC labeled anti-mouse CD25 (eBioscience), fixed, and permeabilized using Fixation & Permeabilization kits (eBioscience), then intracellularly stained with phycoerythrin-labeled anti-mouse/rat Foxp3 (eBioscience). Immunostained cells were analyzed using a Cyan FACS (Dako Cytomation).
RNA isolation and qRT/PCR
Mice sacrificed by CO2
asphyxiation were perfused with ice cold PBS. Pooled spinal cords from 3 MOG peptide, CFA control, or normal control mice were homogenized and stored in RNAlater
solution (Ambion, TX). RNA was isolated using RNeasy Lipid Tissue Mini Kit (QIAGEN, CA) and stored at −80°C. cDNA was prepared using Reaction Ready First Strand cDNA Synthesis kits (SuperArray Bioscience Corp., MD). Real-time PCR was performed using Mouse Toll Like Receptor Signaling Pathway Microarrays and Real-Time SYBR Green PCR Master Mixes (SuperArray Bioscience Corp., MD). To rule out DNA contamination, non-RT (non-reverse transcribed) controls were included for every RNA batch prepared. Additional primer sets were used to verify the microarray results and to examine expression levels of additional genes involved in innate immunity and not included in the arrays (Supplemental Table 1
). The mRNA levels of the assayed genes were normalized to mRNA levels of the housekeeping gene Hsp90ab1.
Spinal cord immunohistology
Mice anesthetized by intraperitoneal administration of ketamine (150mg/kg) and xylazine (16mg/kg) were perfused with PBS, followed by 4% paraformaldehyde (v/v) in PBS. Lumbosacral spinal cords from at least 3 mice on each of days 3, 7, 10, 12, 14, 21, 35, and 98 to 101 post-administration of MOG peptide in CFA, or of CFA alone (both groups also received 2 injections of pertussis toxin as described above) were immunohistologically examined. Paraffin-embedded sections were subjected to antigen retrieval with either sodium citrate, pH 6.0, or citraconic anhydride, pH 7.4 (Alelu-Paz et al, 2008
). The sources for the primary antibodies used for immunohistology, and the antigen retrieval methods (if any) employed with each, are listed in Supplemental Table 2
. Bound antibodies were detected using species and isotype-specific fluorescently conjugated or biotinylated secondary antibodies, and visualized by laser scanning confocal microscopy.
In Thy1-STOP-EYFP/EmxCre double transgenic mice, EYFP expression was visualized in ten µm cryostat cross-sections of L3 spinal cord by immunostaining with a fluorescein-conjugated anti-green fluorescent protein antibody (Rockland). Microscopic fields encompassing both dorsal corticospinal tracts were photographed using a 100× objective mounted on a Nikon laser scanning confocal microscope, and images were tiled together using Nikon NIS-Elements software. All EYFP-labeled axons in the lumbar dorsal corticospinal tract cross-sections prepared from the MOG peptide EAE and CFA control mice were counted with the aid of NIH Image J software.
Data analysis and statistics
Molecular data were derived from at least 3 independent experiments at each time-point. Results of spinal cord qRT/PCR assays were analyzed using the Mann-Whitney U test. Differences between qRT/PCR results obtained from MOG peptide or CFA control spinal cord extracts and those obtained from normal mice of the same age were classified as significant if p<0.05 and the fold difference from normal mice was ≥ 1.5. Immunohistological data were obtained from at least 4 MOG peptide and 4 CFA control mice at each time-point.