Sholl Analysis and Measurement of Microglia Iba-1 Staining Intensity

Scanned images of Iba-1/TMEM119/DAPI-stained 10 μm thoracic spinal cord sections (N = 3–4/mouse, N = 5 mice/group) were opened in Zen (Zeiss). For each mouse, two frames having dimensions of 300 × 150 μm were captured in areas adjacent to sites of submeningeal inflammation in the anterior spinal cord white matter. Iba-1⁺/TMEM119⁺ microglia that had a DAPI⁺ nucleus were manually counted in each frame. The peak intensity of Iba-1 fluorescence for each microglia cell was measured in Zen by placing a circular region of interest (ROI) (5 μm² in area) over each microglia cell and then subtracting the background fluorescence measured when the same ROI was placed adjacent to the cell. Sholl analysis of microglia was performed as follows. Images of individual Iba-1-stained microglia cells that were also verified to be TMEM119⁺ and have a DAPI⁺ nucleus were converted to 8-bit images, opened in Fiji, and then thresholded. Processes from surrounding microglia were deleted manually. The line segment tool was used to draw a line from the center of each soma to the longest process, which provided the maximum process length in μm and provided the region of interest for the Sholl analysis plugin. The Sholl analysis plugin in Fiji (36) was used to measure the intersections at each Sholl radius with the first shell set at 5 μm and subsequent shells set at 2 μm step sizes. This analysis provided the critical radius (radius value from the center of the cell with the highest intersections), the process maximum (the highest number of intersections, regardless of radius value), and the number of primary branches (estimated as the number of primary branches at the first shell). The soma size was measured after manual tracing using the polygon tool. Analyses were performed by an experimenter who was blinded to the identity of experimental groups.