Figure 8

Astrocyte activation and neuronal apoptosis in the brains of WT and db / db mice. (A) qRT-PCR was conducted on RNA extracted from mock- and WNV-infected brains from WT and db/db mice at day 6 and 8 after infection to determine fold-change in GFAP gene expression. Change in the levels of GFAP gene was first normalized to the β-actin gene and then the fold-change in WNV-infected brains was calculated in comparison to the corresponding mock-infected brains. Data represents the mean ± SEM, representing two independent experiments (n = 7 per group). *p <0.05. (B) Cryopreserved brain sections from WNV-infected WT and db/db mice at day 8 after infection were stained for GFAP. Immunoreactivity of GFAP was higher in WNV-infected db/db mice. The photomicrographs demonstrate representative images obtained from two independent experiments (n = 4 per group). Bars, 20 μm. (C) Cryopreserved brain sections from WNV-infected WT and db/db mice at day 8 after infection were stained with H & E. Black arrows identify neurons. (D) TUNEL assay was conducted on cryopreserved brain sections from WNV-infected WT and db/db mice at day 8 after infection to evaluate neuronal apoptosis (Green, white arrows and enlarged inset of the arrowed box). The photomicrographs demonstrate representative images obtained from two independent experiments (n = 4 per group). Bars, 20 μm. (E) Quantitative representation of TUNEL-positive cells from 15 different brain areas per section (total 2 brain sections per mice) from two independent experiments (n = 4 per group). Number of TUNEL-positive cells were significantly higher in the brain of db/db mice. *P <0.05. (F) Co-immunostaining of TUNEL-positive cells with NeuN (neuronal cell marker). The photomicrographs demonstrate representative images obtained from two independent experiments (n = 4 per group). GFAP, glial fibrillary acidic protein; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; SEM, standard error of mean.