Fig. 6From: Protein kinase B (AKT) upregulation and Thy-1-αvβ3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosisIn vivo assay of Cx43 phosphorylation in mouse cortex and in vitro treatment of astrocytes with AKTi to protect neurons from damage. Coronal section of the right (R, Vehicle) and left-brain hemisphere (L, AKTi) of C57BL/6 J male mice injured by the insertion of a needle in both hemispheres. Mice were injected with the vehicle in R (DMSO) or the AKT inhibitor (AKTi, 60 nmol) in L, and killed 24 h or 72 h post-surgery. A Brain tissue sections were labeled with anti-GFAP (green) antibodies, anti-pS373Cx43 (red) antibodies, or DAPI (nuclei, blue) at 72 h. Magnification bar = 50 μm. A threefold digital zoom was applied to the yellow dash square-marked area in the merged picture and appears to the right of the panels. B Brain tissue sections labeled with anti-pS373Cx43 (red) antibodies at 24 h and 72 h. Values in the graphs are the quantification of the mean intensity of GFAP (C) or of pCx43 in arbitrary units (a.u.) (D). E, F Quantification of two different morphological parameters (% differentiation and neurite length) using the NeuroJ software (ImageJ, USA) on bright-field microscopy images. CAD cells were differentiated for 48 h in serum-free medium (SFM) containing sodium selenite or undifferentiated by keeping them in serum-containing medium (SCM). Other conditions included were differentiated CAD cells treated with medium previously conditioned by astrocytes left only in SFM (ACM control), or treated with TNF or with TNF + AKTi for 48 h. These astrocytes were incubated 5 days in SFM to obtain the ACM. Percentage of differentiated CAD cells with processes > 15 µm (E); average length of the processes extended by differentiated cells in microns (F). Values are mean ± s.e.m. from 3 separate experiments, *p < 0.05 (Mann–Whitney test)Back to article page