Drug application Wortmannin, an irreversible PI3K inhibitor, and LY294002, a reversible competitive PI3K inhibitor were purchased from Sigma(St. Louis, MO). Both PI3K inhibitors were dissolved in 1% DMSO.ephrinB1-Fc and EphB1-Fc was purchased from R&D Systems Inc. (Minneapolis, MN) and dissolved in saline. The dosages of drugs are based on the results of preliminary experiments. The usage and dose for each drug were presented in the parts of results and figure legends. The method described by Hylden and Wilcox [49]was used for an intrathecal injection of drugs. In brief, a 28-gauge stainless steel needle attached to a 25-ml Hamilton microsyringe was inserted between the L5 and L6 vertebrae in conscious mice. A sudden slight flick of the tail indicated entry into the subarachnoid space. A volume of 5 ml of drug solution or physiologic saline was injected over a 30-s period into the subarachnoid space, and the injection cannula was left in place for a further 15 s. Motor function was evaluated by observation of placing or stepping reflexes and righting reflexes at 2 min before nociceptive test. Animals with signs of motor dysfunction were excluded from the experiments. Formalin Test The procedure used was essentially the same as that reported by Hunskaar and Hole [50]. Approximately 30 min before testing, mice were individually placed in perspex observation chambers (10620615 cm) for adaptation. Then, the animals were taken out of the chamber, and 10 ml of 1% formalin in 0.9% saline was injected subcutaneously into the dorsal surface of the right hind paw with a 25-ml Hamilton syringe with a 28-gauge needle. Following injection, mice were immediately returned to the observation chamber. The amount of time spent licking and biting the injected paw and the number of flinching paw were measured from 0 to 5 min (the first phase) and from 10 to 40 min (the second phase) after formalin injection and was considered as indicative of nociception. Chronic Constrictive Injury Model Chronic constrictive injury (CCI) model was performed following the method of Bennett and Xie [51]. In brief, mice were anesthetized with sodium pentobarbital (40 mg/kg, intra- peritoneal injection). Left sciatic nerve was exposed at mid-thigh level through a small incision, and a unilateral constriction injury just proximal to the trifurcation was performed with three loose ligatures using a 5-0 silk thread (spaced at a 1-mm interval). In sham-operated animals, the nerve was exposed but not ligated. The incision was closed in layers,and the wound was treated with antibiotics. Measurement of Thermal Hyperalgesia Thermal hyperalgesia was measured using the paw-withdrawal latency (PWL) according to the method described by Hargreaves et al. [52] In brief, mice were individually placed in clear plastic chambers (769611 cm) and allowed to acclimatize to the environment for 1 h before testing. The radiant heat was directed to the plantar surface of each hind paw that was flushed against the glass or site of injection of solution through the glass plate. The nociceptive endpoints in the radiant heat test were the character- istic lifting or licking of the hind paw. The time to the endpoint was considered as the PWL. The radiant heat intensity was adjusted to obtain basal PWL of 12–15 s. An automatic 20-s cutoff was used to prevent tissue damage. Thermal stimuli were delivered three times to each hind paw at 5-min intervals.In CCI model, withdrawal latencies were measured in the same animal on both PI3K & ephrinBs on Spinal Nociceptive Information the ipsilateral (ligated) and the contralateral (unligated) paws. Results were expressed as the difference scores of PWL (s) = contralateral latency-ipsilateral latency. Measurement of Mechanical Allodynia Mechanical allodynia was measured using the paw-withdrawal threshold (PWT) according to the method described by Cristiane F. Villarreal et al. [53] Mechanical hypernociception was evaluated in mice using the Dynamic Plantar Aesthesiometer (Ugo Basile) test. This apparatus has a pressure transducer coupled to a digital force detector that records the applied force in grams. Animals were placed in individual plastic boxes (20625615 cm) on a metal mesh floor and allowed to acclimate for 1 h. The test consists of evoking a hind-paw flexion reflex using a filament containing Universal Tip that touches the plantar surface and exerts an upward force (maximal 50 g) on the plantar surface of the mouse hind paw. The end point was defined by the withdrawal of the paw followed by clear flinching movements. With paw withdrawal, the recorded force was automatically displayed. The results are expressed by the intensity of hypernociception (in grams), Measurements were taken in triplicate and the average was taken as paw withdrawal threshold in grams. In CCI model, withdrawal thresholds were measured in the same animal on both the ipsilateral (ligated) and the contralateral (unligated) paw. Results were expressed as the difference scores of PWT (g) = contralateral threshold-ipsilateral threshold. The behavioral testing was performed by an investigator blinded to the treatment. Immunohistochemistry Mice were anesthetized with sodium pentobarbital (60 mg/ kg,intraperitoneal injection) and were subjected to sternotomy followed by intracardial perfusion with 20 ml saline and 100 ml 4% ice-cold paraformaldehyde in 0.1 mol/l phosphate buffer(PB). The spinal cord of L4–L5 was removed, postfixed in 4% paraformaldehyde overnight, and subsequently allowed to equil- ibrate in 30% sucrose in PB overnight at 4uC. Thirty micrometer transverse series sections were cut on a cryostat and stored in phosphate buffer. After washing in phosphate buffer saline, the tissue sections were incubated in phosphate buffer saline contain- ing 5% normal goat serum and 0.3% Triton X-100 at room temperature for 30 min. For the Fos protein assay, the sections were incubated in primary polyclonal rabbit-anti-Fos antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) at 4uC for 48 h. The sections were then incubated in biotinylated goat anti- rabbit (1:200) at 37uC for 1 h and in avidin-biotin-peroxidase complex (1:100; Vector Labs, Burlingame, CA) at 37uC for 2 h. Finally, the sections were treated with 0.05% diaminobenzidine for 5–10 min. Sections were rinsed in phosphate buffer saline to stop the reaction, mounted on gelatin-coated slides, air-dried, dehydrated with 70–100% alcohol, cleared with xylene, and cover slipped for microscopic examination.For analysis of the change of Fos protein expression, we examined five L4–5 spinal cord sections per animal, selecting the sections with the greatest number of positive neurons.For each animal, we counted the total number of positive neurons in the bilateral spinal cord I–V and X lamina, regardless of the intensity of the staining. Western Blot Analysis The spinal cords of mice were quickly extracted and stored in liquid nitrogen. Tissue samples were homogenized in lysis buffer(pH 7.4) containing (in millimoles): Tris, 20.0 mM; sucro- se,250.0 mM; Na3VO4, 0.03 mM,MgCl2, 2.0 mM, EDTA, 2.0 mM, EGTA, 2.0 mM; phenylmethylsulfonyl fluoride, 2.0 mM; dithiothreitol, 1.0 mM; protease inhibitor cocktail,0.02% PLoS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e40930