Animals and EAE Induction
Female SJL/J were purchased from Jackson Laboratories Inc. (Bar Harbor, ME). Mice were housed in conventional, pathogen-free facility at the New Research Building, Harvard Medical School (Boston, MA). For induction of EAE, SJL/J mice were immunized with 150μg of PLP139-151 (New England Peptide LLC, Gardener, MA, USA) as described previously (Rasmussen et al., 2007
). Clinical disease was assessed according to the following score: 0, no disease; 1, loss of tail tone; 1.5, poor righting ability; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and fore limb weakness; 5, moribund.
The mice were sacrificed at different time points according to the disease phase and clinical score. Acute EAE was defined as peak of disease (around 13 days post immunization, dpi) when mice reached a minimal clinical score of 2; EAE was considered chronic after the first relapse (between 50-60 dpi) when mice reached a minimal score of 1.5. Healthy control mice were sacrificed at least 10 days after immunization with CFA followed by PT.
All mice were housed according to National Institutes of Health guidelines and all experiments were done with the approval of the Animal Care Committee of Harvard University.
Microglia isolation of Subventricular Zone (SVZ) tissue
Mice were deeply anesthetized in a CO2 chamber and transcardially perfused with 30ml PBS. Subventricular zone tissue was dissected from a 2mm block containing the lateral ventricle and the ventricular wall. The tissue was cut into pieces and gently digested using the papain containing Neural Tissue Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Mononuclear cells were isolated by percoll gradient (70%/37%) centrifugation and removed from the interphase, washed and resuspended in PBS containing 2% fetal bovine serum (FBS).
Mononuclear cells from HC, acute and chronic EAE SVZ tissue were labeled with FITC-conjugated anti-CD11b and APC -conjugated CD45 and sorted into a CD11b+/CD45lo microglia population using a FACSAria sorter (BD Bioscience). Microglia cells were lysed and total RNA samples were extracted employing TRIzol (Invitrogen) followed by RNeasy Mini Elute Cleanup (Qiagen).
Microarray analysis and statistical analysis
For microarray analysis, a total of 120 mice were sacrificed to isolate microglia. The SVZ area of 20 mice per group was pooled to sort microglia resulting in one RNA sample per group. Two indepentendly isolated RNA samples per group were submitted for microarray analysis.
RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) and total RNA samples where then labeled and hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix), employing standardized protocols and reagents from Affymetrix. Microarray data from biological replicates were combined and normalized using Bioconductor R. Student’s t-test was used to test for significant differences between gene expression levels of acute EAE vs. control samples, chronic EAE vs. control samples and acute EAE vs. chronic EAE samples. A correction for the false discovery rate (FDR) was not used in this pair-wise comparison. Only genes that were significantly modulated (p value <0.05) and passed a fold change of FC>2.0 were implemented in differential gene expression signatures and considered for further analysis.
Gene Ontology, canonical pathway and functional network analysis
Gene Ontology, canonical pathway and functional network analysis were executed using Ingenuity Pathways Analysis (IPA) tools (Ingenuity Systems, Mointain View, CA). IPA is a curated database of previously published findings on mammalian biology from the public literature. Reports on individual studies of genes in human, mouse or rat were first identified from peer-reviewed publications, and findings were then encoded into an ontology by content and modeling experts.
Each clone identifier was mapped to its corresponding gene in the Ingenuity pathway knowledge base. This knowledge base of pathway interactions is scientifically accurate, semantically consistent, contextually rich, broad in coverage, and up-to-date1
Location mapping and functional analysis were then applied on the so-called focus genes, to identify cellular location and biological functions and/or diseases that were algorithmically significant (Fischer’s exact test) to the data set. For the generation of functional networks, focus genes were used as a starting point. A score was computed for each network, that reflects the negative logarithm of the P that indicated the likelihood of the focus genes in a network being found together due to random chance, where only networks of a score >10 were considered as biologically relevant.
Quantitative real time RT-PCR
Quantitative real-time reverse transcription (RT) PCR, total RNA samples were subjected to cDNA generation using the Applied Biosystems high capacity cDNA Reverse Transcrition Kit. Samples were then subjected to real-time PCR analysis on an ABI 7500 system (Applied Biosystems) under fast PCR conditions. Genes analyzed were detected using commercially available assays (Applied Biosystems). Relative mRNA level was normalized against GAPDH. Gene expression levels resulting form RT-PCR were compared to gene expression levels from microarray to validate the microarray data, where the following genes were picked for validation according to their differential expression or biological relevance to the set: C3, il18bp, lgals1, thra1, insl6, jag1.
Co-immunostaining and Confocal microscopy and Histology
At the appropriate time points, mice were deeply anesthetized in a CO2 chamber and then transcardially perfused with cold PBS. Brains were removed and snap frozen in liquid Nitrogen. Tissues were then stored in −80°C until further use.
Tissues were cut into sections of 25μm thickness in a freezing microtome. For immunostaining, sections containing the SVZ area were fixed with 4% PFA for 10 min, washed with PBS for 15 min, blocked with 8% horse serum in PBS for 1 hours and then incubated over 2 nights with the following antibodies: rat anti CD11b (1:50, BD Biosciences), rabbit anti iba1 (1:200, abcam), goat anti Jag1 (1:50, Santa Cruz Biotechnology) rat anti C3 (1:100, abcam), mouse anti Il18BP (1:100, Stressgen), rabbit anti Galectin-1 (1:100, kindly provided by Dr. GA Rabinovich), goat anti INSL6 (1:50, Santa Cruz Biotechnology) and rabbit anti Htra1 (1:50, abcam). Sections were rinsed and incubated for 1 h with the appropriate Alexa Flour 488 and 594 secondary antibodies (1:500, Mlecular Probes) and mounted on coverslips. Negative control sections for each animal were treated identically, except that the primary antibodies were omitted. The SVZ region was analyzed with a confocal microscope (LSM 510 Laser Scanning Microscope and LSM 3D analysis sorftware, Linuz, Ogdensburg, NY).
For histological assessment, 20μm thick frozen brain sections were stained with hematoxylin and eosin (H&E).