DOI: 10.5061/dryad.j3tx95xhq
title: Data from: Comparisons of quantitative approaches for assessing microglial morphology reveal inconsistencies, ecological fallacy, and a need for standardization

description: Microglia morphology is used as a measure of neuroinflammation and
 pathology, but different methods to quantify microglia morphology are
 frequently employed across neuroscience. For reliable inference, it is
 critical that microglial morphology is accurately quantified and that
 results can be easily interpreted and compared across studies. We applied
 five of the most commonly used ImageJ-based methods for quantifying the
 microglial morphological response to a stimulus to identical
 photomicrographs and isolated microglial cells, which allowed for direct
 comparisons of the specificity and reliability of each method.
description: Tissue samples analyzed in this study were generated from animals
 used in a previous study that examined the role of microglia in sleep
 after an immune challenge. All data used in that study are publicly
 available in the Dryad digital repository. Briefly, male C57BL/6J mice
 were randomly assigned to PLX (Plexxikon 5622 1200 ppm; formulated in
 AIN-76A rodent chow) diet to deplete microglia or control diet (AIN-76A
 rodent chow) for 21 days. On day 21, mice were given an intraperitoneal
 lipopolysaccharide (LPS; <em>E</em>. <em>coli</em>
 0111:B4, Sigma-Aldrich in sterile saline) injection at 0.4 mg/kg in a
 volume of 0.05 ml to induce an inflammatory challenge. Four days post-LPS
 injection, all mice were returned to standard diet and were given 10 days
 for microglia to repopulate. After 10 days of repopulation, all mice were
 given a second LPS injection and maintained on the standard rodent chow.
 Brains were collected at 7 days following the second LPS
 administration. <strong>Perfusion and tissue
 processing</strong> Seven days following the
 second LPS injection, a lethal dose of Euthasol® was administered. Mice
 were transcardially perfused with phosphate-buffered saline (PBS). Brains
 were drop-fixed in 4% PFA for 24 hours and then cryoprotected in
 successive concentrations of sucrose (15%, 30%). Using the Megabrain
 technique, brains were frozen and cryosectioned at 40 µm in the coronal
 plane and were immediately mounted onto slides.
 <strong>Immunohistochemistry</strong>
 Prior to staining, the slides were baked at 56ºC for 3 hours.
 Slides underwent antigen retrieval (sodium citrate buffer PH 6.0 for 100
 minutes). After washing with PBS, PAP pen was applied to slides. Slides
 were incubated in blocking solution (4% Normal horse serum [NHS], 0.1%
 Triton-100 in PBS) for 60 minutes, followed by incubation in primary
 antibody solution (rabbit anti-Iba1; WAKO cat #019919741 at 1:1000
 concentration in 1% NHS, 0.1% triton-100 in PBS) overnight at 4°C. Slides
 were then washed in PBS and 0.1% tween, and were incubated in secondary
 antibody solution (biotinylated horse anti-rabbit IgG (H+L); vector
 BA-1100 at 1:250 concentration in 4% NHS and 0.4% triton-100 in PBS) 60
 minutes at room temperature. Slides were washed in PBS and endogenous
 peroxidases were blocked by incubation in hydrogen peroxide for 30
 minutes. After washing in PBS, ABC solution (Vectastain ABC kit PK-6100)
 was applied for 30 minutes, followed by a PBS wash. 3,3′-Diaminobenzidine
 [from Vector DAB peroxidase substrate kit SK-4100] was applied to the
 slides for 10 minutes. Slides were then placed in tap water and ethanol of
 increasing concentrations (70%, 90%,100%). After treating the tissue with
 Citrosolve, coverslips were applied using dibutyl phthalate polystyrene
 xylene mounting medium. <strong>Imaging
 </strong> Z-stacked photomicrographs were taken
 using a 40 × objective lens on a Zeiss Imager A2 microscope via AxioCam
 MRc5 digital camera and Neurolucida 360 software, with consistent
 microscope settings and Z-stack parameters. Three slices per animal were
 taken from between bregma and lambda and were imaged in the retrosplenial,
 somatosensory, and entorhinal cortices. A total of 669 microglia (randomly
 selected using coordinates and a random number generator) from 225
 photomicrographs (345 microglia from 13 control mice, 324 microglia from
 12 treatment mice) were analyzed. All analyses used the same
 photomicrographs and isolated microglia for direct comparison.
 <strong>Percent coverage photomicrograph
 analysis</strong> The steps followed for percent
 coverage calculations were based on previous studies that used percent or
 pixel coverage techniques to indicate microglial reactivity. Percent
 coverage analysis is often referred to as optical/pixel density or
 intensity of staining/fluorescence. Using ImageJ, raw photomicrographs
 were converted to 8-bit and the ‘subtract background’ function was
 applied. Then the photomicrograph was converted to binary and minimal
 adjustments to the threshold were made so that the binary image best
 represented the raw data, and any processing artifact was filtered out.
 The percentage of the image covered by dark pixels was calculated. Data
 from each mouse included 9 cortical photomicrographs (3 brain slices per
 mouse, 3 photomicrographs per slice). We employed two approaches for
 generating percent coverage data: 1) The percent coverage values for the 9
 photomicrographs were averaged to obtain a single percent coverage value
 per mouse, and 2) each of the 9 separate percent coverage values per mouse
 was retained as individual data points without averaging the
 values. <strong>Full photomicrograph skeletal
 analysis</strong> Iba1 staining was analyzed
 using the skeletal analysis plugin following the protocol previously
 published. In brief, photomicrographs were pre-processed by converting to
 8-bit and applying the FFT bandpass filter in ImageJ. The
 brightness/contrast of the photomicrograph was then adjusted to best
 visualize the branches of the microglia. The unsharp mask was then applied
 to further increase the contrast of the photomicrograph, and the despeckle
 function was applied to remove pixels/noise. The threshold was then
 adjusted, and the despeckle, close, and remove outliers functions were
 applied. The binarized image was then skeletonized. Microglial cell somas
 were counted manually to obtain a total microglial count per
 photomicrograph. The total microglial branch length, branch endpoints, and
 number of branches were calculated across the entire image and then
 averaged by the number of microglial cells per frame.
 <strong>Fractal analysis</strong>
 According to the previously published protocol, randomly selected
 microglia were isolated from the photomicrographs and underwent fractal
 analysis. In brief, this involved converting the photomicrograph to
 binary, creating a region of interest (ROI) that was sized to fit around
 all microglia in the study to ensure the scale of the isolated microglia
 was the same. Using a random number generator, coordinates were used to
 randomly select 3 microglia per image. Using the ROI, each selected cell
 was removed from the binary image. The paintbrush tool was used to remove
 any fragments that were not attached to the cell and connect any branches
 that became fragmented due to image processing, using the original
 photomicrograph as a reference. Once microglia were isolated, the binary
 image was converted to an outline. The FracLac plug-in was used to analyze
 the cells, with ‘box counting’ applied and the ‘grid design Num G’ set to
 4. The convex hull and bounding circle of the cell (image 2D) were
 measured. The fractal dimension (a statistical measure of pattern
 complexity), lacunarity (a geometric measure of how a pattern fills
 space), circularity (how circular the microglial cell is), span ratio
 (longest length/longest width), and density (number of pixels/area) were
 measured. <strong>Single cell skeletal analysis
 </strong> The same cells that were isolated for
 fractal analysis were skeletonized and analyzed with the skeletal analysis
 plugin in ImageJ. The number of branches, the total branch length, and the
 endpoints per microglia were calculated per isolated cell using the
 ‘analyze skeleton function’. The cell body area and perimeter were
 calculated using the multipoint area selection tool.
 <strong>Sholl analysis</strong>
 Sholl analysis was performed in ImageJ on the same isolated cells
 that were used for fractal and skeletal analysis. A radius was drawn from
 the center of the cell body to the end of the longest branch to set the
 upper and lower limit for concentric circle placement. The first circle
 was set as close to the edge of the cell body as possible to ensure the
 cell body was not counted as an intercept on the circle. The distance
 between each circle was set at 5 µm for all cells. The number of times
 that the microglial branches intercepted each of the circles was
 calculated.