experiments in Fig. 5, the cultured microglia (see Supplementary Figure) that had been preincubated with or without ATP (50 mM) were injected intrathecally in normal rats (see Supplementary Methods for full details). Immunohistochemistry Transverse L5 spinal cord sections (30 mm) were cut and processed for immunohistochemistry with anti-P2X4R antibody (Alomone). Identification of the type of P2X 4 R-positive cells was performed with the following markers: for microglia, OX42 (Chemicon) and iba1 (a gift from S. Kohsaka); for astrocytes, GFAP (Boehringer Mannheim); for spinal cord neurons, NeuN (Chemicon) and MAP2 (Chemicon). To assess immunofluorescence staining of cells quantitatively, we measured the immunofluorescence intensity of the P2X 4 R or OX42 as the average pixel intensity within each cell (see also Supplementary Methods). Western blotting Western blot analysis of P2X 4 R expression in the membrane fraction from L4-L6 spinal cord was performed with anti-P2X4R polyclonal antibody (Oncogene) as described in detail in the Supplementary Methods. Microglial culture Rat primary cultured microglia were prepared in accordance with the method described previously 28. In brief, mixed glial culture was prepared from neonatal Wistar rats and maintained for 10-16 days in DMEM medium with 10% fetal bovine serum. Immediately before experiments, microglia were collected by a gentle shake as the floating cells over the mixed glial culture. The microglia were transferred to coverslips or to Eppendorf tubes for subsequent intrathecal administration. Statistics Statistical analyses of the results were made with Student's t-test, Student's paired t-test or the Mann-Whitney U-test.

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.