Quantifying Microglial Morphology and Complexity

A ramified cell is one that has a complicated network of processes that originate from the cell soma. A change in microglia ramification indicates a microglial response to an altered physiologic status, in this case induced by mFPI. ImageJ software (National Institute of Health, https://imagej.nih.gov/ij/^{34 (link)}) and appropriate plugins (i.e. FFT bandpass filter, unsharp mask and close) were consistently used prior to converting all photomicrographs to binary and skeletonized images. In addition to creating skeletonized images, cell somas were manually counted for each photomicrograph. The Analyze Skeleton Plugin (developed by and maintained here: http://imagej.net/AnalyzeSkeleton31) was then applied to the skeleton image which tags skeletal features relevant to microglia ramification: slab voxels (orange, process length) and endpoints (blue). Figure 2 illustrates the workflow process to convert an entire original photomicrograph to a plugin tagged image (original, binary and skeleton); cropped images and an overlay of skeleton to original image is provided for detail and to illustrate that skeletons are representative of the original image. We summarized the number of process endpoints and length from the Analyze Skeleton plugin data output and normalized all data by the number of microglia cell somas in each image to calculate the number of microglia endpoints/cell and microglia process length/cell. In Table 1, we summarize skeleton analysis measures (endpoints and process length/cell) in terms of measure, unit, range, scale, and interpretation.Figure 2

Skeleton analysis of microglia morphologies in Iba1 stained tissue. (a) The process to prepare photomicrographs for skeleton analysis. Original photomicrographs were subjected to a series of uniform ImageJ plugin protocols prior to conversion to binary images; binary images were then skeletonized. An overlay of a resulting skeletonized image (in green) and original photomicrograph shows the relationship between skeleton and photomicrograph. Cropped photomicrographs (below) show additional detail and all skeleton analysis was completed on full sized photomicrographs (Scale bar = 10 µm). (b) The skeletonized images are processed using the Analyze Skeleton plugin (maintained here: http://imagej.net/AnalyzeSkeleton) to identify and tag skeletonized processes as orange, endpoints as blue, and junctions as purple. The tagged data are then organized and data output summarized.

Table 1

Summary of microglia morphology measures.

	Measure	Unit	Range	Scale	Sampling	Interpretation
Process length22	Summed	µm/cell	Continuous	Photomicro-graph	6 photomicrographs/animal	Cell ramification
Process endpoints22	Summed	#/cell	Continuous	Photomicro-graph	6 photomicrographs/animal	Cell ramification
Fractal Dimension36 ,37	\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{regression}\,\mathrm{slope}[\frac{In(N)}{In(\varepsilon )}]$$\end{document}regressionslope[In(N)In(ε)]	DB	1-2	Individual cell	24 cells/animal	Cell complexity
Span Ratio38	\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\rm{convex}}\,{\rm{hull}}\,{\rm{eclipse}}\,{\rm{longest}}\,{\rm{length}}}{{\rm{convex}}\,{\rm{hull}}\,{\rm{eclipse}}\,{\rm{longest}}\,{\rm{width}}}$$\end{document}convexhulleclipselongestlengthconvexhulleclipselongestwidth	Ratio	0-1	Individual cell	24 cells/animal	Cell shape
Density38	\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{\#\,\mathrm{of}\,\mathrm{pixels}\,\mathrm{within}\,\mathrm{cell}\,\mathrm{outline}}{\mathrm{area}\,\mathrm{of}\,\mathrm{convex}\,\mathrm{hull}}$$\end{document}#ofpixelswithincelloutlineareaofconvexhull	\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{\#\mathrm{of}\,\mathrm{pixels}}{{\rm{area}}}$$\end{document}#ofpixelsarea	0-1	Individual cell	24 cells/animal	Cell Size