[30]. In the present study, we hypothesized that like other RTK systems, pain behaviors induced by activation of spinal ephrinBs/ EphBs signaling were mediated by activation of spinal PI3K and we provide strong evidence to support this hypothesis. Results Intrathecal injection of ephrinB1-Fc induced a time- and dose-dependent hyperalgesia and spinal Fos expression In the present study, we found that intrathecal injection of ephrinB1-Fc (0.02, 0.1, and 0.5 mg in 5 ml saline), not control Fc, induced a dose-dependent thermal hyperalgesia and mechanical allodynia in mice, which can last at least up to 24 h and return to baseline level on 48 h after injection of ephrinB1-Fc (P,0.05, from 0.5 to 24 h time point, ephrinB1-Fc 0.1 or ephrinB1-Fc 0.5 group compared with saline or Fc group; fig. 1A). Compared with saline and Fc groups, the calculated area under the curve (22– 48 h) in PWL and PWT tests was significantly decreased in a dose- dependent manner in ephrinB1-Fc group (fig. 1A inset).To rule out a nonspecific effect through the Fc portion, human Fc was used as a control. No significant hyperalgesia was detected after injection of human Fc. these results demonstrated that activation of spinal EphBs receptors could produce thermal hyperalgesia, mechanical allodynia. Although, so far, we still cannot identify which EphBs subgroup was activated dominantly by ephrinB1-Fc. Spinal neuronal sensitization was involved in the development and maintenance of hyperalgesia induced by the different causes. Fos protein, the product of c-fos immediate early gene, has been used as a maker for neuronal activation in the central nervous system [31,32]. There is a positive correlation between the quantity of Fos protein expression and the neuronal activation induced by nociceptive stimuli in spinal cord neurons. To further clarify the algesic effect of intrathecal injection of ephrinB1-Fc, we investigated the change of spinal Fos protein expression following intrathecal injection of different doses of ephrinB1-Fc (0.02, 0.1, and 0.5 mg in 5 ml saline). Intrathecal injection of ephrinB1-Fc, not saline or Fc, induced a dose-dependent increase in spinal Fos protein expression. The expression of Fos protein mainly distributed in I–V lamina of spinal cord (P,0.001, ephrinB1-Fc 0.1 or ephrinB1-Fc 0.5 group compared with saline or Fc group; fig. 1B). These results further confirmed that activation of spinal ephrinBs/EphBs system can sensitize the spinal neurons and induce pain behaviors in na¨ıve mice. Intrathecal injection of ephrinB1-Fc induced a time- dependent and dose-dependent activation of spinal PI3K and AKT Some studies have shown that spinal PI3K activation mediated inflammatory or injury-induced spinal sensitization and hyperal- gesia [21,22,23,24,25,26,27]. Furthermore, PI3K also mediated central sensitization and hyperalgesia induced by activation of central RTK system NGF/TrkA and G-CSF/G-CSFR signaling [22,23,25]. Thus, we want to know if ephrinBs/EphBs signaling, like other RTK pathways such as NGF/TrkA and C-GSF/C- GSFR, is involved in the regulation of the central sensitization through a PI3K-dependent mechanism. To determine whether intrathecal(i.t.) injection of ephrinB1-Fc activates PI3K/AKT pathway, we measured the time course and dose-dependent relationship of expression of PI3K and phosphorylation of downstream kinase AKT (S473) as an indicator of activation of PI3K pathway in the spinal. As shown in fig. 2, i.t. injection of ephrinB1-Fc (0.5 mg) caused an increase of PI3K-p110c on already detectable 10 min after injection and reaching maximal level on 30 min and returning to baseline level on 8 h after PI3K & ephrinBs on Spinal Nociceptive Information injection (PI3K-p110c: P,0.05 at 0.17 h, P,0.01 at 0.5 h, compared with 0 h group; fig. 2A). The same injection increase the expression of phosphorylation of AKT on already detectable 30 min after injection, which reached its peak by 1 h and lasted at least up to 8 h after injection (p-AKT: P,0.05 at 0.5 h, P,0.01 at 1 h, compared with 0 h group; fig. 2B). To further determine ephrinB1-induced activation of PI3K/AKT pathway, we picked up 30-min time point to perform dose–response experiments. I.t. injection of ephrinB1-Fc (0.02, 0.1, and 0.5 mg) produced a dose- dependent increase of spinal PI3K-p110c expression and of phosphorylation of AKT (P,0.05 or P,0.01, ephrinB1-Fc 0.1 or ephrinB1-Fc 0.5 group compared with Fc group; fig. 2C, 2D). To explore the role of PI3K in AKT activation by ephrinB1-Fc, we intrathecally injected wortmannin, an irreversible PI3K inhibitor, or LY294002, a reversible competitive PI3K inhibitor, at 30 min before ephrinB1-Fc injection, then tested the expression of spinal p-AKT at 1 h after ephrinB1-Fc injection. We found that both pretreatments prevented the increase of p-AKT expression (P,0.05, compared with DMSO-ephrinB1-Fc group; fig. 2E). It suggested that PI3K contributed to the activation of spinal AKT by ephrinB1-Fc. In our previous experiments, to exclude a nonspecial effect of the Fc portion, human Fc was used as a control for ephrinB1-Fc. We found that no significant hyperalgesia was detected after injection of human Fc. Therefore, vehicle for ephrinB1-Fc was used as its control in the present study. Inhibition of spinal PI3K prevented and reversed pain behaviors induced by intrathecal injection of ephrinB1-Fc The current data have indicated that intrathecal injection of ephrinB1-Fc induced thermal hyperalgesia and mechanical allodynia, which was associated with activation of spinal PI3K pathway. Therefore, inhibition of PI3K should alleviate ephrinB1- Fc induced pain behaviors in theory. As we expected, pre- treatment with wortmannin (0.016, 0.08, and 0.4 mg in 1% DMSO) or LY294002 (0.2, 1, and 5 mg in 1% DMSO), not DMSO, at 0.5 h before i.t. injection of ephrinB1-Fc (0.5 mg),dose- dependently prevented thermal hyperalgesia and mechanical allodynia induced by i.t. injection of ephrinB1-Fc (0.5 mg). Compared with DMSO-ephrinB1-Fc group, PWL and PWT were significantly elevated in W0.4-ephrinB1-Fc and LY5- ephrinB1-Fc group at 0.5–8 h after i.t. injection of ephrinB1-Fc (P,0.05 or P,0.01) (fig. 3A, 3B). The calculated area under curve (AUC) (22–48 h)was significantly increased in W0.4-,W0.08- ,LY5- or LY1-ephrinB1-Fc group in PWL test, and in W0.4-,LY5- or LY1-ephrinB1-Fc group in PWT test (P,0.05 or P,0.01) (fig. 3A, 3B inset). These results suggested that activation of PI3K pathway was involved in the initiation of pain behaviors induced by i.t. injection of ephrinB1-Fc. Then we asked if PI3K pathway also participated in its maintenance process. To address this question, wortmannin (0.016, 0.08, and 0.4 mg) or LY294002 (0.2, 1,and 5 mg) was administrated at 0.5 h after i.t. injection of ephrinB1-Fc(0.5 mg).We found that post-treatment with both PI3K inhibitors dose-dependently reversed the established thermal hyperalgesia and mechanical allodynia by i.t. injection of ephrinB1-Fc (P,0.05 or P,0.01) (fig. 3C, 3D).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 in PWT test (P,0.05 or P,0.01) (fig. 3C, 3D inset), suggesting that PI3K pathway is required for the maintenance of pain behaviors induced by i.t. injection of ephrinB1-Fc. This effect lasted at least up to 8 h after injection of PI3K inhibitors. PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e40930