Fractal Analysis Using FracLac for ImageJ

For investigating the morphological changes of microglia following ischemic infarct, photomicrographs were acquired from the location (red square in Figures 3A,C,E), 300 μm away from the infarct (peri-infarct cortex), where microglia activation was to be expected high. We utilized our computer-aided morphologic analysis to include fractal analysis (FracLac for ImageJ), which quantifies cell complexity (fractal dimension, fD) (Karperien et al., 2013). Five microglia randomly selected for this analyze within each photomicrograph (6 photomicrographs per animal). A total of 30 cells chosen for fractal analysis (using a grid and random number generator) per animal in each region. The additional structures that abut and surround each cell were eliminated from the analysis by manual deletion using a digitizing tablet and ImageJ. Binary images were then converted to outlines using ImageJ. Fractal dimension (fD) is the assessment of microglia complexity, which quantifies each cell’s contour bounded by the endpoints and process lengths. FracLac for ImageJ calculates the microglia fD for each cell using a box plot protocol that determines the amount of pixel detail with increasing scale, where N = the number of pixels or “detail” at a particular scale (ε) (Figure 5H and Table 1; Morrison et al., 2017). These calculations and relationships were well described in the previous studies (Karperien et al., 2013; Morrison et al., 2017).

Summary of microglia/macrophage morphology measures.

Time course of microglia/macrophage activation after cortical stroke. Representative images of TTC staining and immunostaining of all microglia/macrophages (CD11b) from ischemic core (IC), peri-infarct area, and hippocampus (Hip) coronal sections at 1 (A,B), 2 (C,D), and 7 (E,F) days after 90-min dMCAo in rats. The morphological changes and increasing number of microglia/macrophages were obviously found at 2 days after ischemia. Scale bar is 50 μm (immunofluorescence of CD11b) and 5000 μm (TTC staining).

Complexity analysis of CD11b-positive cell morphologies in the peri-infarct cortex with/without VPA administration. (A) Microglia/macrophages are morphologically dynamic cells able to change form from highly ramified to a completely amoeboid-like shape lacking processes. This transition can be very rapid under pathological conditions. The forms illustrated here represent snapshots of a transformation that is reversible at every time point, with variation with each form shown. (B–G) Complexity analysis of microglia/macrophages in CD11b-stained tissue. The process to prepare photomicrographs for complexity analysis: Original photomicrographs were subjected to a series of uniform ImageJ plugin protocols prior to conversion to binary images. Binary images were then analyzed by using FracLac for ImageJ, which quantifies single cell complexity (fractal dimension, fD). The calculated Df of the cell is shown below its binary image. Representative images of morphological changes in CD11b-staining cells within the sham-operated cortex in vehicle-treated rats (B), sham-operated cortex in VPA-treated rats (C), peri-infarct cortex at 2 days after 90-min dMCAo in vehicle-treated rats (D), peri-infarct cortex at 2 days after 90-min dMCAo in VPA-treated rats (E), peri-infarct cortex at 7 days after 90-min dMCAo in vehicle-treated rats (F), and peri-infarct at 7 days after 90-min dMCAo in VPA-treated rats (G). (H) Illustration of FracLac box counting method to derive fractal dimension calculations of a CD11b-positive cell outline. Shape detail is quantified as scale increase, represented by orange boxes. Box counting equation is summarized in Table 1. (I) Summary data and statistical analysis of fractal dimension at 2 and 7 days after ischemia. Fractal dimension was decreased in the peri-infarct cortex compared with the sham-operated cortex at 2 and 7 days post-stroke. However, VPA treatment restored the fractal dimension of CD11b-positive cells in the peri-infarct cortex at 2 days and 7 days post-stroke. All post hoc analyses are reported in the figure (2 days: ###p < 0.001 vs. sham and dMCAo + vehicle; ***p < 0.001 vs. dMCAo + vehicle and dMCAo + VPA; 7 days: #p < 0.05 vs. sham and dMCAo + vehicle; *p < 0.05 vs. dMCAo + vehicle and dMCAo + VPA). (J) The schemes illustrate graphically the calculation base for the circularity index for the manual analysis. Q22. (K,L) Soma size and circularity index were increased in the peri-infarct cortex compared with the sham-operated cortex at 2 and 7 days post-stroke. However, VPA treatment decreased the soma size and circularity index of CD11b-positive cells in the peri-infarct cortex at 2 and 7 days post-stroke. All post hoc analyses are reported in the figure (2 days: ###p < 0.001 vs. sham and dMCAo + vehicle; ***p < 0.001 vs. dMCAo + vehicle and dMCAo + VPA; 7 days: ###p < 0.001 vs. sham and dMCAo + vehicle; ***p < 0.001 vs. dMCAo + vehicle and dMCAo + VPA). Twenty-five to thirty cells per region of n = 4 rats in each group.