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Regulation of the NR2B-CREB-CRTC1 Signaling Pathway Contributes to Circadian Pain in Murine Model of Chronic Constriction Injury

Xia, Tianjiao PhD, MD; Cui, Yin MD; Qian, Yue MD; Chu, Shuaishuai MD; Song, Jia MD; Gu, Xiaoping PhD, MD; Ma, Zhengliang PhD, MD

doi: 10.1213/ANE.0000000000000991
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Numerous clinical investigations have revealed the circadian rhythm changes in the perception of chronic pain, and most clinical chronic pain types peak in the night. However, it is still undiscovered whether circadian rhythm of pain exists in rodents and the specific mechanism that may underlie it. Our study was conducted to investigate the rhythmic changes of hyperalgesia behavior in a chronic constrictive injury (CCI) model of rodents and to explore the role of the N-methyl-d-aspartate receptor 2B (NR2B)-cAMP response element binding protein (CREB)-CREB-regulated transcription coactivator 1 (CRTC1) signaling pathway in this pain rhythm.

METHODS: A CCI operation was performed to mimic clinical chronic pain. Paw mechanical withdrawal threshold and paw withdrawal thermal latency were used to test pain behavior in rats; a von Frey cilia test was used to test mechanical hyperalgesia in mice at Zeitgeber time (ZT) 4, ZT10, ZT16, and ZT22 for 14 contiguous days. The relative mRNA and protein expression of NR2B, CREB and CRTC1 in the suprachiasmatic nuclei and the dorsal horn were measured by real-time polymerase chain reaction and Western blot. CRTC1 and CREB interference adenovirus vectors were injected intrathecally at 2 time points, respectively (ZT12 and ZT0), to further explore the proper time point for pain treatment.

RESULTS: During the period of chronic pain state, the pain behavior of CCI rodents showed a circadian rhythm with the peak at ZT4 or ZT10 daily. The pain thresholds were significantly different between the activity period and the rest period. The expressions of NR2B, CRTC1, and CREB at the spinal level were consistent with the pain rhythm. The intrathecal treatment with CRTC1 or CREB interference adenovirus from day 7 to day 9 after CCI surgery markedly improved pain behaviors. Nevertheless, when given at ZT0, they were both more effective at relieving peak pain than drugs given at ZT12.

CONCLUSIONS: Pain behavior in the chronic pain of CCI displayed circadian rhythm and was associated with circadian secretion of pain-related receptors. The NR2B-CREB-CRTC1 signaling pathway may play a crucial role in this rhythm. Moreover, our results suggest that measures to relieve pain should be taken before pain reaches its peak.

Published ahead of print October 5, 2015

From the Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department, Nanjing University, Nanjing, Jiangsu, China.

Accepted for publication July 20, 2015.

Published ahead of print October 5, 2015

Funding: This research was supported by National Natural Science Foundation of China (81371207, 81171047, 81070892, and 81171048), Natural Science Foundation of Jiangsu Province (BK2010105), and the Grant from the Department of Health of Jiangsu Province of China (XK201140, RC2011006).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Zhengliang Ma, PhD, MD, Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, 321 Zhong Shan Rd., Nanjing, Jiangsu 210008, P.R. China. Address e-mail to;

© 2016 International Anesthesia Research Society