The microglia expand and exhibit highly branched morphology after antibiotic treatment in allo-HCT mice. (A) Schematic overview of the murine GVHD model. BALB/c mice were treated with antibiotics (daily dose of 1 mg metronidazole, 1 mg vancomycin, 1 mg gentamicin, and 1 mg cefoxitin in 200 μL water by oral gavage) or vehicle for 14 days before and 14 days after transplantation. BALB/c mice were lethally irradiated (split-dose 10 Gy) and transplanted with allogeneic or syngeneic BM (5 × 10⁶ cells) and T cells (3 × 10⁵ cells) from C57BL/6 (allo-HCT) or BALB/c (syn-HCT) donor mice. Organs were analyzed on day 14 after transplantation. (B) Representative images showing IF staining for Iba-1 in the cortex of BALB/c mice transplanted with either syngeneic BM and T cells (upper left), T-cell–depleted (TCD) allogeneic BM only (upper right), or allogeneic BM and T cells. Allo-HCT mice were treated with either vehicle (lower left) or antibiotics (lower right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit immunoglobulin G (IgG; H + L) Alexa Fluor Plus 647 secondary antibody was incubated for 90 minutes at 4°C. Sections were imaged with Zeiss Axio Imager M2m fluorescence microscope with Plan-Apochromat 20×/0.8 M27 objective. Scale bar, 50 μm. (C-D) Scatter dot plots showing numbers of Iba-1⁺ cells per mm² cortex (C) and cerebellum (D). BALB/c mice were transplanted with syngeneic BM and T cells, TCD allogeneic BM only, or allogeneic BM and T cells. Allo-HCT mice were treated with either vehicle or antibiotics as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± standard error of mean (SEM). P values were calculated using ordinary 1-way analysis of variance (ANOVA). (E) Representative images showing IMARIS-based 3-dimensional (3D) reconstruction of the microglia (upper panel) and IF staining for Iba-1 (red) and DAPI (4′,6-diamidino-2-phenylindole; blue; lower panel) in the cortex of GVHD mice treated with either vehicle (right column) or antibiotics (left column). The primary antibody was incubated for 48 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor 568 secondary antibody was incubated for 48 hours at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss LSM710 (Plan-Apochromat 63×/1.4 Oil DIC M27) or Zeiss LSM880 (Plan-Apochromat 63×/1.4 Oil DIC M27) confocal laser scanning microscopes. Scale bar: 5 μm. (F-K) Scatter dot plots showing IMARIS-based semiautomated quantification of the morphological parameter filament dendrite length (F), filament number of terminal points (G), filament number of dendrite branch points (H), filament number of dendrite segments (I), filament dendrite volume (J), and filament dendrite area (K). BALB/c mice were transplanted with allogeneic BM and T cells and treated with vehicle or antibiotics as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test.