The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD-95)/N-methyl-d-aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4-5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2-PSD-95, pSHP2-PSD-95, PSD-95-NR2B, and PSD-95-pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury-induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD-95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5μg/rat for 7days) prevented injury-induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC-87877 (an SHP2 antagonist; 1, 10, or 100μM/rat) and SIRPα1-neutralizing antibodies (1, 10, or 30μg/rat) suppressed spinal nerve ligation-induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2-PSD-95 and PSD-95-NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC-87877 and SIRPα1 antibody treatments, except it prevented the allodynia-associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1-SHP2 interaction exists that subsequently triggers SHP2/PSD-95/NR2B signaling, thereby playing a role in neuropathic pain development.

Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in painrelated neural plasticity at the spinal cord level have been identified. To test whether Src-family tyrosine kinase-dependent glutamatergic N-methyl-D-aspartate receptor NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate pelvic-urethra reflex potentiation, we recorded external urethra sphincter electromyogram reflex activity and analyzed protein expression in the lumbosacral (L6-S2) dorsal horn in response to intrathecal ephrinB2 injections. When compared with vehicle solution, exogenous ephrinB2 (5 g/rat it)-induced reflex potentiation, in associated with phosphorylation of EphB1/2, Src-family kinase, NR2B Y1336 and Y1472 tyrosine residues. Both intrathecal EphB1 and EphB2 immunoglobulin fusion protein (both 10 g/rat it) prevented ephrinB2-dependent reflex potentiation, as well as protein phosphorylation. Pretreatment with PP2 (50 M, 10 l it), an Src-family kinase antagonist, reversed the reflex potentiation, as well as Src kinase and NR2B phosphorylation. Together, these results suggest the ephrinB2-dependent EphBR activation, which subsequently provokes Src kinase-mediated N-methyl-D-aspartate receptor NR2B phosphorylation in the lumbosacral dorsal horn, is crucial for the induction of spinal reflex potentiation contributing to the development of visceral pain and/or hyperalgesia in the pelvic area. pelvic pain; urethra; Src-family kinase; N-methyl-D-aspartate IN THE LUMBOSACRAL DORSAL horn, neurotransmission mediated by glutamatergic N-methyl-D-aspartate receptors (NMDARs) has been implicated in processing nociceptive afferent inputs coming from the lower urinary tract (4, 10). Spinal administration of NMDAR agonists has dose-dependently facilitated the visceromotor reflex, together with pressor responses to pelvic noxious stimulation (14). Conversely, blockage of NMDAR using pharmacological antagonists has inhibited pain behavior caused by irritation of the pelvic viscera (24, 43). Among subunits of NMDAR, the role of the NR2B subunit in pain development has been intensively investigated, as electrophysiological studies have demonstrated that phosphorylation of specific NR2B tyrosine residues is an important determinant for NMDAR-mediated currents (22), which defines the role of NMDARs in pain-related neural plasticity (3,17,18,20). The signaling of Src kinases, a family of protooncogenic tyrosine kinases, has been shown to modulate NMDAR-mediated synaptic transmission and plasticity (1). In inflammatory animal models, the intrathecal administration of Src-family kinase inhibitors prevented spinal NR2B phosphorylation and behavior hyperalgesia, implying that Src-dependent phosphorylation of NMDAR NR2B subunits plays a crucial role in the development of postinflammatory pain and/or hyperalgesia (35).EphB receptors (EphBRs), transmembrane molecules, were initially identified as guidance cues during neural development (40). In the last decade, studies have demonstrated that the intera...