The effects of i.t. injection of Ad-PTEN on PTEN in the dorsal lumbar spinal cord. Fourteen days after i.t.; spinal cord sections (10 μm) are from i.t. injection of vehicle (A), Ad-GFP (B), and Ad-PTEN (C) groups. Immunostaining images of the spinal cord show cells labeled with PTEN (red). Quantification of PTEN (D) immunoreactivity in the ipsilateral dorsal horn of the lumbar spinal gray matter compared with vehicle group. Injection (i.t.) of Ad-PTEN significantly upregulated spinal PTEN immunoreactivity. Each bar in (D) represents the mean ± SEM with six rats per group. Ad-GFP, adenovirus-mediated green fluorescent protein; Ad-PTEN, adenovirus-mediated phosphatase and tensin homolog deleted from chromosome 10. Confocal double-immunofluorescent staining of PTEN (red) with NeuN (neuronal-specific marker; (E), green), GFAP (astrocyte specific marker; (F), green), and OX-42 (microglial specific marker; (G), green) in the dorsal horn region of the lumbar spinal cord of the i.t. Ad-PTEN group. The merged images of (E), (F), and (G) (yellow; white arrow) indicate co-localization of PTEN with NeuN, GFAP, and OX-42 immunoreactive cells in the spinal cord, respectively. The confocal results show that spinal PTEN was primarily co-localized with astrocytes in i.t. Ad-PTEN group. Scale bars are 200 μm for panels A-C and 50 μm for panels E-G. *P < 0.05 compared with the vehicle group.