BACKGROUND: It has been reported that activation and/or translocation of protein kinase C (PKC) is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflammatory pain.OBJECTIVE: To observe PKC changes in the dorsal horn of spinal cord using immunohistochemistry and to measure the time-course during persistent pain produced by chemical stimulation with a right hind-paw injection of formalin. DESIGN: Randomized controlled animal experiment.SETTING: Institute of Basic Medical Science, Hebei Medical UniversityMATERIALS: The present experiment was performed at the Department of Pathophysiology, Institute of Basic Medical Science, Hebei Medical University between September 2000 and June 2002. Forty-two Sprague-Dawley rats, weighing 260-280 g, irrespective of gender, were provided by the Center of Animal Experimentation at Hebei Medical University. PKC antibody was provided by Sigma, USA. Immunohistochemistry kits were purchased from Zhongshan Biotechnology Company, Beijing. HPIAS-1000 definition multicolor system was provided by Qianping Wuxiang Project Company of Tongji Medical University. Animal use during experimentation was consistent with the standards of Animal Ethics Committee.METHODS: Sprague-Dawley rats were divided randomly into control (n = 6) and experimental groups (n = 36). Experimental rats were given an intracutaneous injection of 5% formalin into the planta surface of the right hind-paw. Animals with inflammatory pain were anesthetized and sacrificed to obtain the L5 spinal region at 1, 3, 12 hours, 1, 3, and 7 days after formalin treatment, with 6 rats in each time group. The spinal cords at the L5 region were collected from the control group following sodium chloride injections into the planta surface of the right hind-paw, identical to the experimental group. MAIN OUTCOME MEASURES: Pain reaction of experimental rats after formalin treatment. PKC-positive neurons, and distribution of PKC-immunoreactive particles, in the ipsi- and contralateral dorsal horn were investigated during different stages of inflammatory pain using immunohistochemistry. RESULTS: All 42 rats were included in the final analysis, without any loss. Pain reaction: consistent with previous findings, it was determined that a unilateral injection of formalin into the hind-paw resulted in significant edema and induced a series of nociceptive responses, such as licking, biting, or shaking the injected paw. The maximal inflammation change was observed 1 day after formalin injection and changes did not disappear until the day 7. Number of the PKC positive neurons: results demonstrated that the number of PKC immunoreactive neurons in the dorsal horn increased slightly after formalin injection at 1 hour, compared with the control group. PKC immunoreactivity was up-regulated at day 1, reduced at day 3, and appeared to recover at day 7. The number of PKC-positive neurons in the contralateral side was less than the ipsilateral side at each time sampled. Distribution of PKC immunoparticles over the neurons: PKC immunoreactivity was observed in the nucleus and cytoplasm, as well as on or near the membrane of neurons and synaptosomes in the spinal cord of the control group. PKC activated and translocated from nucleus to the membrane-associated site following formalin treatment. Significant changes were observed at 1 hour and 1 day. The intensity of staining was stronger in the ipsilateral side than the contralateral side at all time points following formalin injection (P < 0.01), whereas the expression patterns of PKC immunoreactivity in the nuclei were very similar in the right and left hemispheres.CONCLUSION: PKC expression in the dorsal horn of the spinal cord peaked at 1 hour and 24 hours, and was very obvious at 24 hours. Protein kinase C expression in the spinal cord increased bilaterally, although it was greater in the ipsilateral hemisphere. In addition, PKC expression at the neuronal membrane and synaptosome was significantly increased. These results indicate that PKC expression is activated in the dorsal horn of the spinal cord during hyperalgesia.