Microglial Morphological Phenotypes in Human dACC

A general assessment of IBA1-immunoreactive (-IR) cells was first conducted to evaluate the relative distribution and abundance of microglial phenotypes in dACC gray and white matter. In all subjects, microglia in the gray matter were generally randomly distributed across and within layers, whereas they appeared to be aligned with myelinated fibers in the adjacent white matter (Figure 1). Four distinct morphological phenotypes were easily recognizable in both gray and white matter. These morphologies corresponded to the previously described microglial phenotypes classically associated with differing states of activation: ramified, primed, reactive, and amoeboid [12 (link),14 (link),15 (link),22 (link),43 (link)]. In human dACC, IBA1-IR cells were categorized using the following distinctive features: ramified microglia displayed a small but defined cell body that appeared spherical in the gray matter (Figure 2a) and ellipsoid in the white matter (Figure 3a). In both cortical compartments, ramified microglia displayed several highly branched processes. Primed microglia in gray matter remained highly ramified, albeit with fewer higher-order branches, but presented a distinctive ellipsoid-like soma (Figure 2b). In the white matter, primed microglia were also highly ramified, but displayed a noticeably wider cell body (Figure 3b). Reactive and amoeboid microglia both presented amoeboid-shaped cell bodies. The processes extended by reactive microglia were less extensive and generally longer than the cell body diameter (Figures 2c and 3c), whereas amoeboid microglia were either devoid of processes or had few unbranched processes seen to be within the length of the cell-body diameter (Figures 2d and 3d).
Having performed a preliminary assessment that revealed very little intra-phenotypic morphological variability between subjects, we proceeded by analyzing the first 10 IBA-IR cells that corresponded unambiguously to the above-described features corresponding to each phenotype. We analyzed a total 40 gray matter and 40 white matter microglia, with 10 cells/phenotype being randomly selected and reconstructed across subjects. On average, 7.4 ± 1.0 cells per subject were traced, reconstructed, and analyzed. Cells were sampled throughout the cortical thickness, but since no noticeable difference was seen between layers, laminar distributions were not recorded. Cells were traced, reconstructed, and their morphometric features characterized as previously described [44 (link)]. In brief, under a 100× (Numerical aperture 1.4) oil immersion objective (Olympus BX51 light microscope, Olympus America Inc., Richmond Hill, On, Canada) processes were analyzed in three dimensions within single sections using a computer-based tracing system (Neurolucida v. 8.10.2, MBF Bioscience, Williston, VT, USA), whereas cell bodies were analyzed in two dimensions (area at its largest cross-sectional diameter). Cell body area, maximum and minimum feret diameter, roundness as well as number, length, branching points (nodes and ends) and volume of processes were measured for each IBA1-IR cell. A spherical cell body is calculated by the ratio between feret diameters. Feret is defined as the distance between two parallel lines drawn tangentially to the cell body; the minimum feret is the shortest chord drawn in the cell body and the maximum Feret is the longest, as shown in the blue and purple lines respectively in Figure 4a. In a spherical cell body, the difference between the maximum and minimum ferets (max-min feret) tends to zero.