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Objective: To observe the complete Freund's adjuvant (CFA)-induced inflammatory pain rat peripheral part of the brain regions at different time points within the central nervous system: the amygdala, hypothalamus midline nuclear group and board kernel base periaqueductal gray matter ( PAG) in astrocytes, microglia markers and cytokine (IL-1β, TNF-α) changes in the expression of the gene and protein levels. Methods: (1) behavioral detection: detection of radiant heat stimulation and mechanical stimulation in rats bilateral the claw at different time points after injection of CFA temperature hyperalgesia and mechanical allodynia changes. (2) RT-PCR: 21 SD rats were randomly divided into seven groups, each group of three. The first group was the control group, the group 2-4 4h after injection of CFA, 3d, 14d group, 5-7 group injected with saline after 4h, 3d, 14d group. Amygdala, hypothalamus midline nuclear group take the rats in each group and the board kernel base, PAG, extraction of total RNA by semi-quantitative RT-PCR analysis of the same part at different time points in each group astrocyte activation markers-of GFAP microglial activation markers-CD14, proinflammatory cytokines IL-1β, TNF-αmRNA expression change. Each lane GFAP, CD14, IL-1β, TNF-α and β-actin or GAPDH bands optical density ratio of GFAP, CD14, IL-1β, TNF-αmRNA relative expression levels. T test after use Stata7.0 statistical software for the the line single factor analysis of variance pairwise comparisons. P lt; 0.05 for significant sector value. (3) Western-blot: 21 SD rats were randomly divided into seven groups, each group of three. The first group was normal control group, the group 2-4 12h after injection of CFA, 3d, 14d group, 5-7 group injected with saline after 12h, 3d, 14d group. Amygdala, hypothalamus midline nuclear group take the rats in each group and the board kernel base, PAG, extract protein using Western-blot analysis of the same part at different time points in each group of GFAP, IL-1β, TNF-α protein expression changes. To each lane of GFAP, IL-1β, TNF-α and β-actin bands optical density ratio of GFAP, IL-1β, TNF-α protein relative expression levels. T test after use Stata7.0 statistical software for the the line single factor analysis of variance pairwise comparisons. P lt; 0.05 for significant sector value. (4) immunohistochemistry 3d group CFA and saline rats amygdala: with fluorescent single subject of observation, the hypothalamic midline nuclei and board kernel group, PAG astrocyte marker GFAP and the microglia marker object Iba expression change. Results: (1) prior to injection, the rats to mechanical stimuli flinching reaction threshold value is an average of 11.00 ± 1.32g, 4 hours after injection CFA hyperalgesia reaction threshold value of 7.33 ± 0.42 g (n = 6, P LT; 0.05 VS before injection), 12 hours after injection, reached a peak (1.00 g, p lt; 0.001 VS before injection), this mechanical hyperalgesia continued until day 21, 6 (gp lt; 0.01 VS injection). Before the injection of CFA, rats flinching thermal stimulation incubation period of 10.32 ± 0.74s, the sensitivity peaked 12 hours after the injection, flinching incubation period is 3.18 ± 0.24s (n = 6, p lt; 0.001 VS injections cit.) Inflammatory pain and thermal hyperalgesia sustained to 14 days returned to normal. (2) all brain regions of GFAP mRNA in the expression of the amount of injection of CFA after 3d, 14d and the normal group compared there is significant with increased, CD14, of IL-1β, of TNF-α in the injection of CFA after 4h, 3d, 14d have significantly increased. (3) various brain regions GFAP protein expression in the injection of CFA after 3d, 14d increased IL-1β, TNF-α, 12h, 3d, 14d were increased. (4) GFAP fluorescent single standard, PAG, hypothalamus, amygdala astrocytes in 3d group CFA group than the saline group significantly increase. Iba fluorescent single marked microglia in various brain regions compared to the 3d group CFA with the saline group, the number did not significantly increase proliferation and hypertrophy, but the morphology. Conclusion: microglia activation may be associated with inflammatory pain starting astrocytes may be related to the maintenance of inflammatory pain, increased expression of cytokines may be the occurrence of hyperalgesia plays an important role.
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