Stereological Approaches to Microglia Morphometry

Morphometry of microglia (MG) is an irreplaceable component in the characterization of the multiple types and states of this key cellular element of the brain and spinal cord. Cell number, cell body size and shape, and number and length of MG processes are defining parameters that adopt different values in different neural locations, sexes, and species and change substantially during development and aging and in response to neural trauma, inflammation, or degeneration. This makes MG morphometry a target particularly hard to tackle, both at the single cell and the population level, especially when MG proliferates, migrates, and creates areas of heavily intertwined labeled profiles. In this chapter, we present the application of several stereological methods to the quantification of those parameters on MG identified by Iba-1 immunofluorescence (IF) and immunohistochemistry (IHC). The workflow of the several models of labeling and tissue preparation of MG-containing histological material and cell culture is summarized, and the stereological procedures are detailed for each model. In a nutshell, in IHC-processed sections studied under bright-field microscopy in a NewCAST® setup, the optical disector or fractionator is applied to estimate N and NV, and isotropic virtual planes are used to estimate the LV and LN of MG processes. In confocal images from IF sections, the optical disector or fractionator is again applied to estimate N and NV, and the total vertical projection method as originally designed to estimate the total length of a bounded linear feature in three dimensions is applied, with a minor tweak, to estimate the LV and LN of MG processes. The cell body size and number of primary processes are also estimated on this material. Finally, two-dimensional stereological methods are applied to monolayer MG cell cultures to estimate cell body size and circularity. Some of these procedures were applied for the first time in recent studies by the authors.