Amoebida

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.

Negative staining was performed on the fixed supernatant from co-culture. We deposited 5 μL onto the glow-discharged grid for 20 min at room temperature. The dried grid was contrasted with a small drop of 1% ammonium molybdate for 10 seconds and the grid was then observed on a Tecnai G20 (FEI, Germany).
For the infectious cycle description, a pure viral suspension was used to infect four flasks of 30 mL, each containing amoeba suspension of 5.10⁵ amoebas/mL, at a multiplicity of infection (MOI) of 10. After 30 min of post infection incubation, the amoeba monolayer was washed three times with PAS buffer to eliminate non-internalized viruses. This time point was designated as H0. A total of 10 mL of the infected cultures were distributed into new culture flasks incubated at 30 °C. A culture flask containing a non-infected flask of amoeba was used as the negative control. At 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, and 28 h post-infection (hpi), each culture flask corresponding to a specific time point was centrifuged at 720× g for 10 min, and the pellets were fixed for the transmission electron microscopy procedures. With regards to these, A. castellanii-infected cells were recovered and pelleted for 10 min at 5000× g. The pellet was re-suspended in 1 mL of phosphate-buffered saline (PBS) with 2% glutaraldehyde–0.1 M cacodylate and incubated for at least 1 h at 4 °C. Each pellet was then washed three times with 0.1 M cacodylate–saccharose and resuspended in the same buffer. After re-pelleting, each sample was then embedded in Epon resin by using the following standard method: 1 h of fixation in 1% osmium tetroxide, two washes in distilled water, dehydration in increasing successive ethanol concentrations (30%, 50%, 70%, 96%, and 100% ethanol), and embedding in Epon-812. Ultrathin (70 nm) sections were post-stained with 5% uranyl acetate and lead citrate [14 (link)]. Electron micrographs were obtained on a Tecnai G20 F20 TEM (FEI, Germany) operated at 200 keV. ImageJ (https://imagej.nih.gov/ij/) software was used to determine particle size at the different time points of the cycle.

Morphometric analysis was performed by an investigator that was unaware of the identity of the experimental groups. The number of Iba1-immunoreactive cells was estimated with the optical disector method in the hilus of the dentate gyrus of the hippocampus, using total section thickness for disector height at 40× [39 (link),40 ] and a counting frame of 220 × 220 μm. Section thickness was measured using a digital length gauge device (Heidenhain-Metro MT 12/ND221; Traunreut, Germany) attached to the stage of a Leitz microscope. A total of 28 counting frames were assessed per animal. In addition, the percentage of Iba1 immunoreactive cells with different morphologies was also assessed. Cells were classified in five morphological types: Type I, cells with few cellular processes (two or less); Type II, cells showing three to five short branches; Type III, cells with numerous (>5) and longer cell processes and a small cell body; Type IV, cells with large somas and retracted and thicker processes and Type V, cells with amoeboid cell body, numerous short processes and intense Iba1 immunostaining. For each animal, 120 cells were analyzed in the hilus of the dentate gyrus of the hippocampus.

IHC was performed according to our previous report [57 (link)]. The antibodies that were used are listed in Additional file 1: Table S1. Images were processed using the Zen image acquisition software package (Carl Zeiss, Oberkochen, Germany) and ImageJ software. The soma area was evaluated using images of Iba1 staining, and the amoeboid score was calculated by the following formula: (soma area/entire Iba1-stained area) × 100 (%). The Iba1- and CD68-costained areas were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).