To analyze microglial morphology at the single-cell level, we used the “FracLac plugin” in ImageJ (https://imagej.net/ij/plugins/fraclac/FLHelp/Introduction.htm) after we collected the binary images of each microglial cell (93). Here, we use the parameters including fractal dimension (D), lacunarity, convex hull area (CHA), convex hull perimeter (CHP), cell circularity (CC), and convex hull span ratio to assess the complexity and circularity of microglial cells. Fractal D is the method for discerning various microglial shapes, ranging from simple round forms to intricate branched structures (94, 95). In this study, box-counting software was used to enumerate the number of boxes encompassing any foreground pixels within the outlined images, which were progressively processed on grids of diminishing calibers. Lacunarity indicates variations in the soma and additional morphological features. This parameter quantifies shape heterogeneity, both in terms of translation and rotation invariance (95). Lacunarity is computed using the box-counting software FracLac, representing the pixel mass distribution in microglia images. CHP is the single outline of the convex hull expressed in microns. CHA refers to the measurement of the area enclosed by the smallest convex polygon, which
