Contribution of Spinal PKCγ Expression to Short- and Long-lasting Pain Behaviors in Formalin-induced Inflamed Mice

Abstract

Background: Over-expression of spinal protein kinase C γ (PKCγ) contributes to the induction of persistent bilateral hyperalgesia following inflammatory injury, yet the role of spinal PKCγ in short- and long-lasting pain behavior is poorly understood. Objective: This study aimed to characterize the contribution of spinal PKCγ to spontaneous pain and long-lasting bilateral hyperalgesia in formalin-induced inflamed mice using pharmacological inhibition. Study Design: Laboratory animal study. Setting: The study was performed in the Department of Human Anatomy and K.K. Leung Brain Research Centre, Preclinical School of Medicine, the Fourth Military Medical University (Xi’an, China) and the Department of Anesthesiology, Fuzhou General Hospital (Fuzhou, China). Methods: Male mice were unilaterally intraplantarly injected with formalin to induce inflammatory pain. Spontaneous pain behaviors, including flinches and lickings, were recorded by off-line video during the first hour post-injection and counted. Using von Frey tests, longlasting bilateral mechanical paw withdrawal thresholds were determined before injection and at indicated time points thereafter. Temporal expression of spinal PKCγ was observed by immunohistochemical staining. For pharmacological inhibition, mice were treated daily with intrathecal Tat carrier or selective PKCγ inhibitor KIG31-1, from 1 hour prior to 10 days after formalin injection. Spontaneous pain behaviors and long-lasting bilateral mechanical hyperalgesia were assessed. Spinal PKCγ expression was also observed by using immunohistochemical staining and western blot. Results: The number of PKCγ-immunoreactive (ir) spinal neurons was significantly higher at 10 days, but not 2 hours, after formalin intraplantar injection, and accompanied by long-lasting bilateral hyperalgesia. Furthermore, long-lasting bilateral hyperalgesia could be reversed by pharmacological inhibition of over-expressed spinal PKCγ; however, pretreating with intrathecal KIG31-1 showed no antinociceptive effects on short-term spontaneous pain behaviors. Limitations: All results were obtained from the mice and no PKCγ inhibitors were available through clinical practice. Therefore, it remains difficult to draw definitive connections between animal research and human application. Conclusion: Our findings suggest that spinal PKCγ plays a predominant role in long-lasting bilateral hyperalgesia, but not in the spontaneous pain behaviors induced by formalin. Key words: Formalin, spontaneous pain, mechanical hyperalgesia, protein kinase C gamma, KIG31-1, mice