말초 신경손상으로 유도되는 물리적 이질통 형성에 대한 척수 별아교 세포 내 sigma-1 수용체의 역할

학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2015. 2. 이장헌.BACKGROUND: Chronic pain, such as peripheral neuropathic pain, can be characterized by sensory disorders that include mechanical allodynia (MA, lowering of response threshold to light tactile stimuli) and thermal hyperalgesia (TH, an increased response to a noxious thermal stimulus). The precise mechanisms in the spinal cord underlying the development of MA and TH remain to be clearly defined. The role of sigma non-opioid intracellular receptor 1 (Sig-1R) in modulating central sensitization associated with the development of neuropathic pain has recently been investigated. Moreover, it has been recognized that spinal Sig-1Rs play an important role in the induction of MA in neuropathic pain. However, it was not clearly demonstrated the specific mechanism related to this spinal Sig-1R under the development of MA in neuropathic pain condition. OBJECTIVES: The present study was designed to investigate: 1. the precise cellular location of Sig-1Rs in the spinal cord and whether Sig-1R activation mediates phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK), among mitogen-activated protein kinase (MAPK) signaling pathways. The effects of p38 and ERK inhibitor on the Sig-1R induced pain behaviors were also investigated. 2. the histological and physiological relationships among Sig-1R, p-p38 and glial activation and whether the inhibition of Sig-1R or p38 modulates astrocyte activation in chronic constriction injury (CCI) mice. I also investigated whether this Sig-1R modulation of astrocyte activation is associated with the increase of D-serine in the spinal cord dorsal horn. 3. whether the inhibition of Sig-1R or p38, which modulates astrocyte activity is associated with MA development in CCI mice. In addition, the role of D-serine in the induction of Sig-1R-mediated MA was also investigated in CCI mice. MATERIALS AND METHODS: Primary astrocyte-enriched cell cultures were prepared from newborn C57BL/6 mice. Other experiments were performed on male ICR mice (20-25g). A CCI of the common sciatic nerve was performed according to the method described by Bennett and Xie. For intrathecal injection, I used the modified method of direct transcutaneous intrathecal injection on mice described by Hylden and Wilcox. Sensitization to innocuous mechanical stimulation (MA) was examined using von Frey filaments with forces of a 0.16 g. To assess nociceptive responses to heat stimuli, sensitization to noxious heat stimulation (TH) was examined with a hot-plate apparatus or Hargreaves apparatus. In the present study, the Sig-1R agonist, PRE-084 (3 nmol), its antagonist, BD-1047 (100 nmol), a ERK inhibitor, PD98059 (3, 10, 30 nmol), a p38 inhibitor, SB203580 (0.1, 0.3, 1, 3, 10 nmol), an astroglial metabolic inhibitor, fluorocitrate (0.003, 0.001, 0.03 nmol) and a serine racemase inhibitor, LSOS (1, 3, 10 nmol) were used. Immunohistochemistry and Western blot assay were performed according to each experiment procedure. The computer-assisted image analysis system (Metamorph) was utilized throughout whole experiments. RESULTS: 1. The expression of Sig-1Rs was significantly increased in astrocytes on day 3 following CCI surgery in mice. In cultured astrocytes, Sig-1R expression was also found, and the treatment of PRE-084 increased phosphorylation of ERK and p38, but not JNK. While intrathecal pretreatment with PD98059 inhibited both PRE-084-induced MA and TH, SB203580 dose-dependently inhibited PRE-084-induced MA, but not TH induction was not affected. Intrathecal injection of PRE-084 into naïve mice time-dependently increased the expression of p38 phosphorylation (p-p38), which was blocked by pretreatment with BD-1047. 2. The activation of astrocyte and microglia were increased in the spinal cord on day 3 following CCI surgery in mice. Sustained intrathecal treatment with the BD-1047 during the induction phase (postoperative days 0 to 3), attenuated CCI-induced increase in activation of astrocyte, but not microglia. The number of p-p38-ir astrocytes and neurons, but not microglia was also significantly increased after CCI surgery. Interestingly, intrathecal BD-1047 attenuated the expression of p-p38 selectively in astrocytes but not in neurons. Moreover, intrathecal treatment with a p38 inhibitor attenuated the GFAP expression. The level of D-serine and Serine racemase (Srr) expression were significantly increased in the spinal cord after CCI surgery. D-serine was localized in astrocyte and accumulated around neuron. Srr was also found in astrocyte. The increased level of D-serine was attenuated by sustained intrathecal treatment with BD-1047. Accordingly, Srr expression was also reduced by BD-1047 treatment and I found Srr expression was located on the same cell with Sig-1R-ir cells. 3. Sustained intrathecal treatment with the BD-1047 combined with fluorocitrate synergistically reduced the development of MA, but not TH. Moreover, intrathecal treatment with a p38 inhibitor combined with fluorocitrate synergistically blocked the induction of MA. In addition, sustained intrathecal treatment with the Srr inhibitor reduced the development of MA, and the blockade by BD-1047 treatment was reversed by exogenous D-serine. CONCLUSIONS: The present study demonstrates that the direct activation of spinal Sig-1R significantly increased p-p38 and the activation of spinal p38 was closely involved with the Sig-1R-mediated MA, but not TH. In CCI mice, spinal Sig-1R expression are increased in astrocytes, and that blockade of Sig-1Rs inhibits the pathologic activation of astrocytes via modulation of p-p38, which ultimately prevents the development of MA. The present study also indicates that the Sig-1R-mediated MA is developed by an increase of Srr expression, which in turn causes an increase of D-serine level in the spinal cord of CCI mice. Collectively these findings imply that the increase of p-p38 and D-serine induced by spinal Sig-1R plays an important role in the development of MA in neuropathic pain and further suggest that MA development could be clinically controlled by the Sig-1R modulation.BACKGROUND 1 OBJECTIVES 10 INTRODUCTION 11 MATERIALS & METHODS 17 1 Animal preparation 17 2 Primary astrocyte cultures 17 3 Intrathecal drug injection 18 4 Mechanical allodynia assay 19 5 Thermal hyperalgesia assay 20 6 Western blotting analysis 21 7 Immunohistochemistry 22 8 Image analysis 24 9 Statistical analysis 26 RESULTS 28 1 Cellular distribution of spinal Sig-1Rs and related MAPKs phosphorylation 28 2 The histological and physiological relationships among Sig-1R, p-p38 and astrocyte activation in the spinal cord of CCI mice 41 3 Releasing factor induced by spinal Sig-1R in CCI mice 55 4 Effects on CCI-induced MA and TH 67 DISCUSSION 76 SUMMARY 87 REFERENCES 89 ABSTRACT IN KOREAN 103Docto