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Fig. 6 | Journal of Neuroinflammation

Fig. 6

From: Protein kinase B (AKT) upregulation and Thy-1-αvβ3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosis

Fig. 6

In 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)

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