Schematic representation microglia isolation from the SVZ and microarray strategy. Microglia were isolated from RR EAE during the acute and chonic disease phase (red arrows) and from CFA injected healthy control mice, followed by dissection of the SVZ with flow cytometry sorting of CD11b/CD45 dim microglia. Subsequent microarray and data analysis was performed after 2 rounds of RNA amplification.

IPA analsys of Gene Ontology. (a) Pie-diagram of the cellular location of genes from each up- and downregulated signiture set for acute, chronic and common EAE, with each number representing the number of genes in a given cellular location. (b) Gene Ontology functions of up-regulated genes with location in extracellular space and plasma membrane for acute, chronic and common EAE. Top 5 significant GO functions categorized into “Molecular and Cellular Functions”, Physiological System Development and Functions” and “Diseases and Disorders” are displayed as pie-diagrams with each number representing the number of molecules associated with the given function. (c) Top 3 canonical pathways associated with upregulated genes of secreted and plasma membrane expressed molecules for the signature sets in common EAE, acute EAE, and chronic EAE and their dynamics in each disease phase. Displayed are the significance (−log(p-value); blue bars; left y-axis) and the ratio (number of signature genes present in the canonical pathway / total number of genes associated with the canonical pathway; orange line; right y-axis). The red lines show the threshold for significance (−log (p=0.05))

The microarray data was validated by RT-PCR, correlating the Log2 ratio of the microarray genes with the Log2 ratio of given genes as measured by quantitative RT-PCR. The data show a good linear fit with a correlation coefficient of 0.78 and a slope of 1.18.

Comparison of the significant genes identified as differentially expressed in acute EAE, chronic EAE and acute and chronic EAE. (a) Identification of 1024, 28 and 111 upregulated genes for acute, chronic and common EAE respectively. (b) Identification of 147, 14 and 90 downregulated genes for acute, chronic and common EAE respectively.

(a) Top scoring network for signature genes of secreted and plasma membrane expressed molecules in acute EAE, many of which are involved in neural- or oligodendroglial precursor function, neuroprotection, neurite outgrowth or angiongenesis. (b) High scoring network with an enrichment of complement factors. Nodes represent genes, with their shape representing the functional class of the gene product and edges indicate the biological relationship between the nodes. Genes that were selected as target genes for microarray validation are highlighted with red borders.