PI3K & ephrinBs on Spinal Nociceptive Information Figure 2. Intrathecal(i.t.) injection of ephrinB1-Fc induced a dose- and time-dependent increase of spinal PI3K-p110c and p-AKT expression. The expression of spinal PI3K-p110c and p-AKT was assayed at 0, 0.17-h, 0.5-h, 1-h, 4-h, and 8-h time-points in the time-dependent experiments and at 0.5-h time-point in the dose-dependent experiments after ephrinB1-Fc injection. PI3K inhibitor wormannin (0.4 mg) or LY294002 (5 mg) or DMSO was intrathecally injected at 30 min before i.t. ephrinB1-Fc (0.5 mg),the expression of p-AKT in spinal was assayed at 1 h after ephrinB1-Fc injection in PI3K mediated spinal AKT activation experiment. (A, B) The representative bands (top) for the expression of PI3K-p110c or p- AKT in spinal at different time points after injection of ephrinB1-Fc (0.5 mg) and the quantitative data (bottom) for the expression of PI3K-p110c or p- AKT. The fold change for the density of PI3K-p110c or p-AKT normalized to GAPDH or AKT for each sample, respectively. The fold change of PI3K- p110c or p-AKT level in 0 time point groups was set at 1 for quantifications. *P,0.05, **P,0.01, compared with 0 time point group, n = 4 mice in each group. (C, D) The representative bands (top) for the expression of PI3K-p110c or p-AKT in spinal after injection of various dose of ephrinB1-Fc (0.02, 0.1, and 0.5 mg). The quantitative data (bottom) for the expression of PI3K-p110c or p-AKT. The fold change for the density of PI3K-p110c or p-AKT normalized to GAPDH or AKT for each sample, respectively. The fold change of PI3K-p110c or p-AKT level in Fc groups was set at 1 for quantifications. *P,0.05, **P,0.01, compared with Fc group, n = 4 mice in each group.(E) The representative bands (top) and the quantitative data (bottom) for the expression of spinal p-AKT. The fold change for the density of p-AKT normalized to AKT for each sample. The fold change of p-AKT level in DMSO- saline group was set at 1 for quantifications.*P,0.05, compared with DMSO-ephrinB1-Fc group, n = 4 mice in each group. doi:10.1371/journal.pone.0040930.g002 EphBs receptors were involved in activation of spinal PI3K and AKT in the neuropathic pain model We also observed the similar antinociceptive effect when EphB1-Fc was injected in neuropathic pain model. Consecutive intrathecal injection of EphB1-Fc on 1 h before CCI and days 1, day3 after CCI (0.5 mg for each injection) prevented the induction of thermal hyperalgesia and mechanical allodynia and this effect continued at least for 7 days after CCI (P,0.05 or P,0.01, EphB1-Fc-CCI group compared with Sal-CCI group; fig. 6A). On day 5 after CCI, a peak time point of pain behaviors, L4–L5 spinal cord of some mice were extracted for measurement of Fos protein, PI3K-p110c and p-AKT expression. Injection of EphB1-Fc significantly inhibited the increased expression of spinal Fos protein (P,0.01 compared with Sal-CCI group; fig. 6B), PI3K- p110c and p-AKT expression (PI3K-p110c:Sal-CCI group compared with Sal-Sham group: P,0.01; EphB1-Fc-CCI group compared with Sal-CCI group: P,0.01;p-AKT: Sal-CCI group compared with Sal-Sham group: P,0.01; EphB1-Fc-CCI group compared with Sal-CCI group: P,0.05; fig. 6C) induced by CCI. Intrathecal injection of a single dose of EphB1-Fc (0.5 mg/5 ml) on PLoS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e40930