Lysophosphatidic acid receptor (LPA 1 ) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal (i.t.) injection of LPA evoked a dose-and time-dependent downregulation of myelin-associated glycoprotein (MAG) in the DR through LPA 1 receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA 1 receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund's adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR. Keywords lysophosphatidic acid; neuropathic pain; myelin-associated glycoprotein; dorsal root; calpain; demyelination Lysophosphatidic acid (LPA) is a bioactive lipid mediator that exerts diverse physiological and pathophysiological functions through its cognate LPA receptors (LPA 1-5 and P2Y5) (Noguchi et al. 2009). Recently, we demonstrated that LPA 1 receptor signaling initiates neuropathic pain following peripheral nerve injury, using mice lacking the lpa 1 gene (Lpar1 −/− ) (Inoue et al. 2004, Ueda 2008. Regarding the molecular bases, LPA upregulates pain-related gene expression, such as Ca 2+ channel α2δ-1 subunit and ephrinB1 in the dorsal root ganglion (DRG) and protein kinase C γ-isoform in the spinal cord (Inoue et al. 2004, Uchida et al. 2009). Moreover, LPA causes demyelination of the dorsal root (DR) * Correspondence to: Dr. Hiroshi Ueda, Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan, Tel: +81-95-819-2421; Fax: +81-95-819-2420; ueda@nagasaki-u.ac.jp. † These authors contributed equally to this study.
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Materials and methods
Animals and surgeryMale mice lacking the lpa 1 gene (Lpar1 −/− ) (Contos et al. 2000) and wild type C57BL/6J mice weighing 20-24 g were used. They were kept in a room with a temperature of 21 ± 2°C with free access to standard laboratory diet and tap water. All procedures were approved by the Nagasaki University Animal Care Committee and complied with the recommendations of the International Association for the Study of Pain (Zimmermann 1...