S-Space College of Veterinary Medicine (수의과대학) Dept. of Veterinary Medicine (수의학과) Theses (Ph.D. / Sc.D._수의학과)
Study for the mechanism underlying spinal astrocyte sigma-1 receptor-mediated GluN1 phosphorylation in the development of neuropathic pain
- 수의과대학 수의학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2016. 8. 이장헌.
- Damage to the peripheral nerves can lead to the development of neuropathic pain, which causes considerable suffering and distress to these patients and is associated with several diagnostic symptoms including mechanical allodynia (MA, sensation of pain to non-noxious mechanical stimuli) and thermal hyperalgesia (TH, increased pain response to noxious thermal stimuli). The development of peripheral neuropathic pain involves a variety of pathophysiological mechanisms in both the peripheral and central nervous systems. In this regard, spinal N-methyl-D-aspartate (NMDA) receptors have been shown to play a key role in the development of central sensitization, a phenomenon in which nociceptive inputs to the dorsal horn increase the excitability and synaptic efficacy of neurons in spinal pain pathways. In our previous study, activation of spinal sigma-1 receptors (Sig-1Rs) contributes to the functional potentiation of NMDA receptors via increases in phosphorylation of GluN1 subunit (pGluN1). However, the precise cellular mechanisms underlying Sig-1Rs-related neuropathic pain are not investigated.
The present study was designed to investigate whether: (1) neuronal nitric oxide synthase (nNOS)-induced nitric oxide (NO) mediates Sig-1R-induced increase in spinal pGluN1 expression in pain hypersensitivity and activation of Sig-1R results in a time-dependent modification of nNOS activity in the dorsal horn
(2) Sig-1R modulation of astrocytic D-serine plays an important role in increasing pGluN1 expression via nNOS activation and that this pathway contributes to the development of persistent neuropathic pain induced by peripheral nerve injury
and finally (3) spinal Sig-1Rs modulate the production of reactive oxygen species (ROS) via activation of NADPH oxidase 2 (Nox2) as a downstream of nNOS/NO signaling, ultimately leading to the development of chronic neuropathic pain.
All experiments were performed on Male ICR mice and Sprague-Dawley rats. Neuropathic pain was produced by chronic constriction injury (CCI) of the common sciatic nerve according to the method described by Bennett and Xie with a minor modification. Sensitization to innocuous mechanical stimulation (mechanical allodynia, MA) was examined using the 0.16 g or 2.0 g von Frey filament, and sensitization to noxious heat stimulation (thermal hyperalgesia, TH) was examined with the hot-plate apparatus or plantar analgesia meter. In the present study, the following drugs were used: PRE084 (a Sig-1R agonist), BD1047 (a Sig-1R antagonist), L-NAME (a non-specific NOS inhibitor), 7-nitroindazole (a specific nNOS inhibitor), ODQ (a sGC inhibitor), cyclosporin A (a calcineurin inhibitor), NMDA (a NMDA receptor agonist), D-serine, LSOS (a Srr inhibitor), DAAO (an endogenous D-serine degrading enzyme), fluorocitrate (an astrocyte metabolic inhibitor), chelerythrine (a PKC inhibitor), PKI (a PKA inhibitor), NAC (a ROS scavenger) and apocynin (a NADPH oxidase inhibitor). All drugs were administrated intrathecally. Immunohistochemistry, DHE staining, NADPH-diaphorase staining, NO detection, Western blot assay and co-immunoprecipitation were performed according to each experiment procedure. The image analysis and statistical analysis were performed using a computer-assisted image analysis system (Metamorph) and a Prism 5.0 (Graph Pad Software), respectively.
Intrathecal (i.t.) injection of Sig-1R agonist, PRE084 significantly evoked mechanical and thermal hypersensitivity, and increased the number of PKC-dependent pGluN1-ir cells in spinal cord. The PRE084-induced hypersensitivity and increase in PKC-dependent pGluN1 expression were significantly blocked by pretreatment with L-NAME or 7-nitroindazole. I.t. administration with PRE084 time-dependently decreased the ratio of phosphorylated nNOS (pnNOS) to nNOS expression and the number of spinal pnNOS-ir cells. This decrease in the pnNOS form was prevented by BD1047, a Sig-1R antagonist and cyclosporin A, a calcineurin inhibitor, but not by a sGC inhibitor.
I.t. administration with the D-serine degrading enzyme, DAAO attenuated the facilitation of NMDA-induced pain behaviors induced by PRE084. Exogenous D-serine facilitates NMDA-induced nociception and increases PKC-dependent pGluN1 expression, which was attenuated by preteatment with the nNOS inhibitor, 7-nitroindazole. In CCI mice, i.t. administration with exogenous D-serine during the induction phase of neuropathic pain restored MA and PKC-dependent pGluN1 suppressed by BD1047. Furthermore, administration with the serine racemase inhibitor, LSOS or DAAO suppressed CCI-induced MA, pGluN1, nNOS activation and NO production. I.t. administration with 7-nitroindazole or the sGC inhibitor, ODQ also attenuated CCI-induced MA and pGluN1. By contrast, D-serine and nNOS signaling had no effect on CCI-induced TH or GluN1 expression.
Sig-1R-induced pain hypersensitivity was dose-dependently attenuated by pretreatment with the ROS scavenger, NAC or the Nox inhibitor, apocynin. PRE084 also induced an increase in Nox2 activation and ROS production, which were attenuated by pretreatment with BD1047, apocynin, or 7-nitroindazole. CCI-induced nerve injury produced an increase in Nox2 activation and ROS production in the spinal cord, all of which were attenuated by i.t. administration with BD1047 during the induction phase of neuropathic pain. Furthermore, administration with BD1047 or apocynin reversed CCI-induced increase in pGluN1 expression and MA during the induction phase, but not the maintenance phase.
The present study demonstrates that Sig-1R modulation of astrocytic D-serine plays an important role in potentiating NMDA receptor function via increase in PKC-dependent phosphorylation of GluN1 subunit and that this pathway contributes to the development of persistent neuropathic pain. In addition, nNOS activation mediates the role of D-serine and has pro-oxidant effects in the CNS via modulation of Nox2 activity and concomitant ROS production, leading to modulate persistent pain. Collectively, these findings demonstrate that spinal Sig-1R activation plays a key role in the development of neuropathic pain induced by peripheral nerve injury, and Sig-1Rs could be a useful therapeutic target for alleviating mechanical allodynia under the neuropathic pain conditions.