The Effects Of Adrenomedullin On Astrocyte And Microglia In Vitro

Background:It has been established that nerve injury in the central and periphery nevous systemes activate glial cells. The material basis of sensory signals transmitted from peripheral sensory neurons to spinal cord is the pronociceptive mediator. Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and has been identified to be a pain neuropeptide. Based on the early studies, we hypothesized that AM could activate glial cells. To test this hypothesis, we first set up the technique to obtain high purity of glial cells. Then explore the implication of AM in activation of glial cells in vitro, concerning on cellular morphological changes and the level of activity markers. Finally, we explored whether AM can still exert these effects on glias following the blockade of AM receptors.Methods:Primary cultures of glial cells were prepared from SD rats born in 2-3 day. AM was used to induce the activation of glial cells. Then antagonist and siRNA for RAMP2 were used to confirm the role of AM receptors in inflammatory response of glial cells activated by AM. We used immunocytochemistry and real-time quantitative PCR techniques to examine the changes in cell morphology and function of glial cells.Results:1) Microglial cells and astrocytes were cultured successfully and purity was up to 90%.2) After 3 days-treatment with AM 1 ?M, Microglia cell processes withdraw, and cell body become larger. We further showed that the AM receptor components RAMP2 mRNA and cell-surface receptor TLR4 and P2X4 mRNA increased significantly following exposure of AM. AM receptors antagonist AM22-52 and specific siRNA oligonucleotide for RMAP2 prevent AM induced morphologic and functional changes, which suggest that the AM induced microglia activitation is mediated by AM receptors.3) Astrocytes are treated with AM at 10 nM,100 nM and 1 ?M for 3 d. We found that after incubated with AM 10 nM, somatas of some astrocytes began to grow bigger and round, the number of morphological changed cells increased and fluoresence intensity of GFAP enhanced over time. The effects of AM on astrocytes were higher as the increasing AM concentrations. Subsequent real-time quantitative PCR results showed that AM increased the GFAP mRNA and nducible enzyme COX-2 mRNA expression levels in astrocytes.AM receptors antagonist AM22-52 prevent AM induced morphologic and functional changes. However, AM (10nM) did not significantly affect levels of CCL2 mRNA.Conclusion:These results indicate that AM can directly activate glial cells mediated via the AM receptors.