Gene therapy of pain by expressing endogenous opioid peptide

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For aiming at gene therapy of pain by expressing endogenous opioid peptide, we achieved three main projects in 2002-2003. First, to clarify the significance of fos expression in the spinal cord of rats after the formalin test, we investigated the time course and location changes of c-fos and FOS expression, by using Northern blotting and immunohistochemical technique. The behavior test showed typical two phases induced by the formalin test. The amount of c-fos increased at 0.5 h after the formalin injection and returned to control level at 6 h. Also, the expression of FOS increased at 0.5 h, and the increase continued more than 6 h. At the early phase, the upregulation of FOS occurred in the superficial layers(laminae I-II ), and later it occurred in the deep layers (laminae V-VI) of the dorsal horn. In this study, we demonstrate that the pain caused by formalin injection induces c-fos mRNA translated to FOS protein in the spinal cord neurons. And the location changes of FOS expression … More in the later phase would imply the change of the mechanism of the central sensitization on the formalin test. Second, we investigated the spatial and temporal patterns of Fos expression in the dorsal horn of the spinal cord caused by plantar master in the rat and the effects of pretreatment with local anesthetics(0.5% Bupivacaine, 2% lidocaine). Pain behavior and Fos expression in the L_4-L_5 spinal cord were assessed at 1, 3, 6, 24, 48, 72, and 120 h after the master. The withdrawal threshold to mechanical stimulation decreased significantly at 1 h until 120 h, and pretreatment with local anesthetics increased the threshold significantly at 1 h and 6 h in comparison to that in the saline group. In the saline group, Fos expression was detected predominantly in laminaes I-II and V-VI, and the total Fos expression was maximal at 1 h and then decreased gradually. Pretreatment with local anesthetics suppressed Fos expression significantly in all layers and this suppression continued for several days. This study provides evidence of spatial and temporal changes in Fos expression induced by plantar master. Furthermore, although pretreatment with local anesthetics suppresses Fos expression for several days in the postoperative period, the analgesic effect is observed only for the expected duration of the local anesthetic used. Third, we confirmed that GFP(green fluorescent protein) was produced in spinal cord cells by injection of adenovirus with GFP to rats. Less