Characterization of the BBB during GVHD. Lethally irradiated B6.Csf1r-eGFP × DBA2xF1 or B6D2D1 mice received 5 × 10⁶ TCD BM with or without 0.5 × 10⁶ enriched splenic T cells on day 0. Brains were subsequently assessed at day 14 or day 70 after transplant. (A) Representative confocal images of hippocampal astrocytes and quantification of GFAP⁺ stained area as a percentage of total image area at day 14 after transplant; n = 4 to 7 mice per group; data pooled from 3 independent experiments. (B) mRNA expression of Gfap detected by qRT-PCR at day 14 after transplant; n = 6 mice per group; data representative of 2 independent experiments. (C) Representative confocal images of hippocampal astrocytes and quantification of GFAP stained area as a percentage of total image area at day 70 after transplant; n = 9 to 17 mice per group; data pooled from 3 independent experiments. (D) mRNA expression of Gfap detected by qRT-PCR at day 70 after transplant; n = 4 mice per group; data representative of 2 independent transplants. (E-H) Representative confocal images of VCAM1 expression: (E) hippocampus at day 14, (F) cortex at day 14, (G) hippocampus at day 70, and (H) cortex at day 70. Quantification of VCAM1⁺ stained area represented as a percentage of total image area is shown. White arrows indicate positive staining; day 14, n = 7 to 9 mice per group; data pooled from 3 independent experiments; day 70, n = 5 to 6 mice per group; data pooled from 2 independent experiments. (I-L) Representative confocal images of CD31 expression: (I) hippocampus at day 14, (J) cortex at day 14, (K) hippocampus at day 14, (L) cortex at day 70. Quantification of CD31⁺ stained area represented as a percentage of total image area and quantification of total CD31 fluorescence intensity (arbitrary fluorescence units [AFU]) are shown; day 14, n = 4 to 8 mice per group; data pooled from 3 independent experiments; day 70, n = 4 to 5 mice per group; data from 1 experiment. (A,C,E-H,I-L) Original magnification ×20; scale bar, 50 μm. Nuclei counterstained with DAPI in all images. Significant differences calculated with Mann-Whitney unpaired nonparametric t test. Data are presented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean.