Minocycline Effects on IL-6 Concentration in Macrophage and Microglial Cells in a Rat Model of Neuropathic Pain

Abstract

Background: Evidence indicates that neuropathic pain pathogenesis is not confined to changes in the activity of neuronal systems but involves interactions between neurons, inflammatory immune and immune-like glial cells. Substances released from immune cells during inflammation play an important role in development and maintenance of neuropathic pain. It has been found that minocycline suppresses the development of neuropathic pain. Here, we evaluated the analgesic effect of minocycline in a chronic constriction injury (CCI) model of neuropathic pain in rat and assessed IL-6 concentration from cultured macrophage and microglia cells.

Methods: Male Wistar rat (n=6, 150-200 g) were divided into three different groups: 1) CCI+vehicle, 2) sham+vehicle, and 3) CCI+drug. Minocycline (10, 20, and 40 mg/kg) was injected one hour before surgery and continued daily to day 14 post ligation. Von Frey filaments and acetone, as pain behavioral tests, were used for mechanical allodynia and cold allodynia, respectively. Experiments were performed on day 0 (before surgery) and days 1, 3, 5, 7, 10, and 14 post -injury. At day 14, rats were killed and monocyte-derived macrophage from right ventricle and microglia from lumbar part of the spinal cord were isolated and cultured in RPMI and Leibovitz's media, respectively. IL-6 concentration was evaluated in cell culture supernatant after 24 h.

Results: Minocycline (10, 20, and 40 mg/kg) attenuated pain behavior, and a decrease in IL-6 concentration was observed in immune cells compared to CCI vehicle-treated animals.

Conclusion: Minocycline reduced pain behavior and decreased IL-6 concentration in macrophage and microglial cells.

Keywords: Interleukin-6; Macrophages; Microglia; Minocycline.

Fig. 1

Immunocytochemistry. Immunophenotyping of microglial cells isolated from the lumbar part of rat’s spinal cord using immunocytochemical assay with OX42 monoclonal antibody against CR3/CD11 microglial cell marker. OX42 antibody reaction in free microglial cells isolated from A) sham-operated and C) CCI vehicle-treated rats. OX42 antibody reaction in microglial cells was isolated from B) sham-operated and D) CCI vehicle-treated rats. CCI vehicle-treated animals showed CR3/CD11 marker expression (magnification ⊆40).

Fig. 2

Pain behavior. Paw withdrawal threshold in response to von Frey filaments (A) and the frequency of paw withdrawal in response to acetone (B) before surgery and at different time points after surgery in CCI vehicle-treated, sham-operated and CCI minocycline-treated groups. Minocycline (M, 10, 20 and 40 mg/kg) was injected i.p. Data were analyzed using the analysis of variance (ANOVA) and were presented as means±S.E.M for six rats in each group. Asterisks (**P<0.01 and ***P<0.001) indicate a statistically significant difference between post surgery days and day 0.

Fig. 3

The IL-6 concentration in immune cells. IL-6 concentration of macrophage cells (A) and microglial cells (B) in CCI vehicle-treated, sham-operated and CCI minocycline-treated rats on day 14 post ligation. Data were analyzed using the analysis of variance (ANOVA) and were presented as means±S.E.M for six rats in each group. Asterisks (*P<0.05, **P<0.01 and ***P<0.001) indicate a statistically significant difference when compared to CCI vehicle-treated rats. M, minocycline (10, 20 and 40 mg/kg)