The effects of i.t. Ad-PTEN on CCI-induced upregulation of TNF-α and phospho-mTOR in spinal astrocytes. Spinal cord sections (10 μm) obtained 14 days post-surgery from sham-operated plus i.t. vehicle (A, D), CCI plus i.t. Ad-GFP (B, E), and CCI plus i.t. Ad-PTEN (C, F) groups. Confocal double-immunofluorescent staining of TNF-α (red; A-C) or phospho-mTOR (red; D-F) with GFAP (astrocyte specific marker; green) in the dorsal horn region of the lumbar spinal cord. The merged images (yellow; white arrow) indicate co-localization of TNF-α or phospho-mTOR with GFAP immunoreactive cells in the spinal cord. The confocal results showed that spinal TNF-α and phospho-mTOR were primarily co-localized with astrocytes in the CCI group, whereas i.t. Ad-PTEN significantly attenuated CCI-induced upregulation of spinal TNF-α and phospho-mTOR immunoreactivity in astrocytes. Scale bars are 50 μm for all images.