Effects of human TBI serum anti-GFAP autoantibody primary rat glial cells.

(A–E) Primary rat glia cells were plated on cover slip and cell chamber and incubated in media that included 1/50 serum collected from either human TBI serum with no detectable anti-GFAP autoantibody (Control serum) (A,B) or human TBI serum with strong anti-GFAP autoantibody (C,D). Cells were treated with either vehicle (DMSO) (A,C) or 20 μM A23187 (calcium ionophore) (B,D) for 24 hours, After the 24 h incubation, media was removed and the cells were fixed, stained with Hoechst and developed with anti-human IgG secondary antibody, Scale bars 10 microns. Yellow arrowheads indicate glial cell body staining with IgG while blue arrows indicate glial processes staining. (E) To test of effects of glial health, we used pooled 4 control samples and 9 TBI samples with strong autoantibody response. Cells were co-treated with either vehicle (DMSO) or 20 μM A23187 for 24 hours. Cell death (by LDH release) was measured using CytoTox-Glo™ Cytotoxicity Assay. Luminescence was read and media from each of the different conditions was used to measure the background, which was subtracted from the individual readings. * (P<0.0001) Represents significant increase of glial death in cells treated with TBI serum as compared to cells treated with control serum (control or with A23187, respectively), using an ANOVA analysis with Bonferroni multiple comparisons.