Pain Rhythm Related Molecular Mechanisms In Neuropathic Pain

Background:Numerous clinical investigations revealed that chronic pain changes according to the circadian rhythm,which is usually exacerbated at night.The circadian rhythm is depending on the endogenous circadian clock.This clock is controlled by a set of conserved and well-characterized clock genes,the mRNA and protein levels of which comprise the clock oscillator to generate the circadian rhythm.Suprachiamatic nucleus of the hypothalamus is the central oscillator of the circadian clock.It can drive daily rhythms in biochemistry,physiology and behavior by regulating other oscillators.Expression of clock genes in intumescentia lumbalis of spinal cord,which modulate pain signals,is associated with the rhythm of pain behavior.Sensitization of nociceptive neurons in spinal dorsal is involved in pain.NR2B receptor of the neurons is essential for induction and maintenance of central sensitization,and it can regulate downstream molecules CRTC1 and CREB.CRTC1,as the co-stimulatory molecules of CREB,can cascade up-regulate the phosphorylation of CREB.Recent research revealed that the circadian rhythm in suprachiasmatic nucleus can be directly regulated by NR2B-CRTC1-CREB signaling pathway.Therefore,we investigated the association of the circadian rhythms of neuropathic pain and NR2B-CRTC1-CREB signaling pathway.Purpose:1.To explore the association of murine clock gene expression in spinal cord with suprachiasmatic nuclei during the daily circadian rhythm,and to observe the influence of neuropathic pain on clock genes and "sleep-wake" rhythm.2.To study the pain rhythm change in neuropathic pain.3.To reveal the mechanism of NR2B-CRTC1-CREB signaling pathway in mice spinal neurons in vitro.4.To discuss clinical diagnosis and treatment of chronic pain based on the association between NR2B-CRTC1-CREB signaling pathway and neuropathic pain rhythm.Methods:CCI(Chronic constriction injury)model was established to induce neuropathic pain in mice and rats.Primary neurons of mice spinal cord were cultured in vitro and stimulated by glutamate,which could activate CREB signaling pathway.CREB and CRTC1 interference gland viral vectors was prepared to intervene CCI mice at the alternated spot of day and night,and change of pain behavior and NR2B-CRTC1-CREB signaling pathway was observed.Hyperalgesia behavior was evaluated with paw withdrawal mechanical threshold and paw withdrawal thermal latency in mice and rats.Change of"sleep-wake rhythm" related Gross motor was full real-time monitored by Minimitter.Viability of neurons was detected by CCK8 kits after drug intervention;mechanism underlying the circadian rhythm of neuropathic pain was investigated by Immunofluorescence,Western Blot and real-time quantitative PCR.Results:1.mRNA expressin of Cry1,Per2,Bmall and Clock in spinal cord and PBMCs is correlated with central oscillator in suprachiamatic nucleus of the hypothalamus at daily circadian rhythms.The expression of Cry1,Per2,Bmall and Clock in spinal cord display an obvious oscillates rhythm in CCI mice.2.The "sleep-wake rhythm" in CCI mice is unaffected compared with WT mice.Gross motor movements decrease compared with sham operation group.And paw withdrawal mechanical threshold and paw withdrawal thermal latency in CCI mice exhibit circadian rhythmicity,with a high level during night while low during day time.3.NR2B and CREB of primary spinal cord neurons is activated by glutamate with an optimum concentration of 100?M in vitro,while CREB and CRTC1 innterference adenoviral vectors can inhibit mRNA expression of related clock genes.4.Alterations of NR2B,CRTC1 and CREB in intumescentia lumbalis of spinal cord show a remarkably consistent with the pain rhythm in CCI mice.Inhibitions of CRTC1 and CREB can significantly reduce pain behaviors of CCI mice.Intervention at the spot of day and night can regulate pain rhythm more efficiently.Feedback regulation of NR2B-CRTC1-CREB signaling pathway is correlated with improvement of pain behaviors.Conclusion1.The circadian rhythm of clock genes in spinal cord is correlated with central oscillator in suprachiamatic nucleus of the hypothalamus,expression of clock genes in spinal cord exhibit oscillate rhythm in neuropathic pain.2.Neuropathic pain has an obvious pain rhythm which is characterized with hypnalgia,and "sleep-wake" rhythm was left uninfluenced.3.NR2B-CRTC1-CREB signaling pathway directly regulates the circadian rhythm of neuropathic pain.Inhibition of the pathway can improve Hyperalgesia.