PI3K & ephrinBs on Spinal Nociceptive Information thermal hyperalgesia (top) and mechanical allodynia (bottom) by ephrinB1-Fc (0.5 mg).Compared with ephrinB1-Fc-DMSO group, the calculated area under the curve (AUC) (22–8 h) (inside bar figure) was significantly increased in ephrinB1-Fc-W0.4-, -W0.08, –LY5,or –LY1 group in PWL test, and in ephrinB1-Fc-W0.4, -W0.08,or –LY5 group in PWT test, *P,0.05, **P,0.01 compared with ephrinB1-Fc-DMSO group, n = 8 mice in each group. doi:10.1371/journal.pone.0040930.g003 the fifth day after CCI reversed the established thermal hyperalgesia and mechanical allodynia, and this effect could last up to 8 h (P,0.01,CCI-EphB1-Fc group compared with CCI-Sal group; fig. 6D). Spinal Fos protein (P,0.01 compared with CCI- Sal group; fig. 6E), PI3K-p110c and p-AKT (PI3K-p110c:CCI- Sal group compared with Sham-Sal group: P,0.05; CCI-EphB1- Fc group compared with CCI-Sal group: P,0.05;p-AKT: CCI- Sal group compared with Sham-Sal group: P,0.05; CCI-EphB1- Fc group compared with CCI-Sal group: P,0.05; fig. 6F) expression detected on 2 h after injection of EphB1-Fc also were markedly inhibited. PI3K activity is required for ERK activation induced by intrathecal injection of ephrinB1-Fc Previous and recent studies have shown that both peripheral and spinal ERK activation was involved in hyperalgesia induced Figure 4. Inhibition of PI3K partially inhibited or reversed the increased expression of spinal Fos protein by i.t. ephrinB1-Fc. PI3K inhibitor wortmannin (0.4 mg) or LY294002 (5 mg) or DMSO was intrathecally injected at 30 min before and after i.t. ephrinB1-Fc (0.5 mg). Spinal Fos protein expression was assayed at 2 h after ephrinB1-Fc injection. (A,C) Representative Fos immunohistochemical staining and (B,D) the quantitative data of Fos expression in the spinal cord of mice in pre-treatment experiments or post-treatment experiments. ###P,0.001, compared with Sal- DMSO or DMSO-Sal group, *P,0.05, compared with DMSO-ephrinB1-Fc or ephrinB1-Fc-DMSO group, n = 6 mice in each group, Scale bar = 100 mm. doi:10.1371/journal.pone.0040930.g004 PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e40930