Glial Activation And Proinflammatory Cytokine Expression In The Brain Regions Induced By Peripheral Inflammation Pain

Objective: To observe the activation of astrocytes, microglia and expression of proinflammatory cytokines in the supraspinal brain regions (the PAG, the midline and intralaminar thalamic nuclei and amygdala ) by CFA-induced peripheral inflammation. Method: (1) Behavioral testing: using thermal and mechanical stimulation to observe hyperalgesia and allodynia of rats at different time point after injection of CFA into unilateral hindpaw. (2) RT-PCR: twenty-one SD rats were randomly divided into 7 groups, 3 rats in each group. One group served as normal control and the others served as CFA-injection 4h, 3d and 14d group, saline-injection 4h, 3d and 14d group, respectively. Then PAG, the midline and intralaminar thalamic nuclei and amygdala were isolated and total RNA was extracted. Semi-quantitative reverse transcriptase -polymerase chain reaction (RT-PCR) method was used in detection of the expression of GFAP, CD14, IL-1βand TNF-αmRNA in every brain area. The relative expression level of GFAP, CD14, IL-1βand TNF-αmRNA was indicated by the ratio of the optical density value of GFAP, CD14, IL-1βand TNF-αto that of GAPDH orβ-actin. The data was analyzed with ANOVA followed by t test using the Stata 7.0 software. p < 0.05 was considered statistically significant. (3) Western Blot: twenty-one SD rats were randomly divided into 7 groups, 3 rats in each group. One group served as normal control and the others served as CFA-injection 12h, 3d and 14d group, saline-injection 12h, 3d and 14d group, respectively. Then PAG, the midline and intralaminar thalamic nuclei and amygdala were isolated and protein was extracted. Western Blot method was used in detection of the expression of GFAP, IL-1βand TNF-αprotein in every brain area. The relative expression level of GFAP, IL-1βand TNF-αprotein was indicated by the ratio of the optical density value of GFAP, IL-1βand TNF-αto that ofβ-actin. The data was analyzed with ANOVA followed by t test using the Stata 7.0 software. p < 0.05 was considered statistically significant. (4) Immunohistochemistry: using immunofluorescence to observe the expression of GFAP and Iba in PAG, thalamus and amygdala 3 days after CFA-injection and saline-injection. Results: (1) Before injection, the paw withdrawal threshold was 11.00±1.32g, Four hours later, it decreased to 7.33±0.42g on the ipsilateral paw of the CFA-treated rats( n = 6, p < 0.05 ). The markedly decrease was shown at 12h after CFA injection, and it was 1.00g ( p < 0.001 ). This mechanical allodynia lasted to 21days. For the thermal test, the paw withdrawal latency (PWL) was 10.32±0.74s before injection. The hyperalgesia peaked at 12 h and PWL was 3.18±0.24s ( n = 6, p < 0.001). It was recovered on Day 14. (2) The expression of mRNA for GFAP was increased at day 3 and day 14 after CFA injection, and the expression of mRNA for CD14, IL-1βand TNF-αwas up-regulated at 4 hours, day 3 and day 14 following CFA injection at every tested brain region. (3) The expression of protein for GFAP was increased at day 3 and day 14 after CFA injection and the expression of IL-1βand TNF-αwas enhanced at 4 hours, day 3 and day 14 following CFA injection at every tested brain region. (4) Immunohiatochemistry revealed that intraplantar administration of CFA produced a significant increase in the expression for GFAP in PAG, thalamus, amygdala after 3 days of inflammation, compared with the control group. And the Iba-positive cells in these brain regions had no significant change in number, but appeared hypertrophia after 3 days of inflammation, compared with the control group. Conclusion: The activation of microglia and astrocytes may be involved in the initiation and maintenance of inflammatory pain, respectively. Enhanced cytokines expression might have a role in eliciting behavioral hypersensitivity.