PI3K & ephrinBs on Spinal Nociceptive Information Figure 1. Intrathecal (i.t.) injection of ephrinB1-Fc produced a dose- and time-dependent hyperalgesia and spinal Fos protein expression. Paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded at 1-h, 2-h, 4-h, 8-h, 24- h, and 48-h after ephrinB1-Fc injection. Spinal Fos protein expression was assayed at 2-h after ephrinB1-Fc injection. (A)Intrathecal injection of 0.1 or 0.5 mg ephrinB1-Fc, not 0.02 mg ephrinB1-Fc or 0.5 mg control Fc, produced thermal hyperalgesia and mechanical allodynia. 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. Inset figures showed that the calculated area under the curve (AUC) (22–48 h) in paw-withdrawal latency (PWL) and paw-withdrawal threshold (PWT) test was significantly decreased in a dose-dependent manner in ephrinB1-Fc group. *P,0.05, **P,0.01 compared with saline or Fc group. n = 8 mice in each group. (B) Representative immunohistochemical staining and the quantitative data of Fos expression in the spinal cord of mice. Intrathecal injection of ephrinB1-Fc (0.02, 0.1, and 0.5 mg), not saline or Fc (0.5 mg), induced a dose-dependent increase in spinal Fos protein expression in superficial lamine of ipsilateral spinal cord,*** P,0.001 compared with saline group, n = 6 mice in each group. Scale bar = 100 mm. doi:10.1371/journal.pone.0040930.g001 Inhibition of spinal PI3K prevented and reversed spinal Fos protein expression induced by intrathecal injection of ephrinB1-Fc The expression of Fos protein also may be a useful tool to examine the effectiveness of different analgesic regimens [31,32]. To further clarify the analgesic effect of inhibition of PI3K on pain induced by i.t. injection of ephrinB1-Fc, we investigated the effect of pre-treatment or post-treatment with PI3K inhibitors on spinal Fos protein expression induced by i.t. injection of ephrinB1-Fc (0.5 mg). DMSO, wortmannin (0.4 mg) or LY294002 (5 mg) was intrathecally administrated at 30 min before or after i.t. injection of ephrinB1-Fc (0.5 mg) and spinal Fos protein expression was assayed at 2 h after ephrinB1-Fc injection. The results showed that both pre-treatment and post-treatment with both PI3K inhibitors partially inhibited the spinal Fos expression induced by i.t. injection of ephrinB1-Fc (P,0.05, compared with DMSO- ephrinB1-Fc or ephrinB1-Fc-DMSO group; fig. 4). EphBs receptors were involved in activation of spinal PI3K and AKT in the inflammatory pain model Spinal ephrinBs or EphBs signaling played a critical role in the development and maintenance of pathological pain. To test whether spinal PI3K and AKT activation contribute to the role of ephrinBs or EphBs signaling in inflammatory pain, we analyzed the effect of intrathecal injection of EphB1-Fc, which can block ephrinBs or EphBs pathway, on expression of spinal PI3K-p110c and p-AKT in inflammatory models (fig. 5). Pretreatment with intrathecal injection of EphB1-Fc (0.5 mg/5 ml) at 30 min before formalin injection significantly reduced the time of licking and the number of flinching hind paw in phase I and II responses (EphB1- Fc-Formalin group compared with Sal-Formalin group, P,0.05 at phase I and II; fig. 5A). The spinal Fos protein (P,0.01 compared with Sal-Formalin group; fig. 5B), PI3K-p110c and p-AKT (PI3K- p110c: Sal-Formalin group compared with Sal-Sal group: P,0.05; EphB1-Fc-Formalin group compared with Sal-Formalin group: P,0.05;p-AKT: Sal-Formalin group compared with Sal-Sal group: P,0.01; EphB1-Fc-Formalin group compared with Sal- Formalin group: P,0.05; fig. 5C) expression, detected at 2 h and 30 min after injection of formalin, respectively, also was markedly inhibited. PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e40930