| ObjectBrain asymmetry is thought to be one of the biological characteristics in humans and non-human primates, which is evidenced on the basis of anatomical, neurochemical and functional data. Brain lateralization is involved in the regulation of physiological systems including the immune systems,two sides of the brain are differently involved in the modulation of immune responses as demonstrated by lesional and behavioral approaches. In the physiological condition the right neocortex depresses immune functions, whereas the left neocortex enhances immune functions. Brain lateralization has effect on the immune system through hypothalamic-pituitary-adrenal(HPA) axis.Microglia are typically described as the resident macrophage occupying the parenchyma of the CNS,after stimulation, miacroglia become immune effector cells, which are main sources of cytokines in the CNS, although astrocytes can secret cytokines, microglia are more responsible for LPS than astrocytes. The LPS receptors of microglia including CD14,TLR4 and MD-2. Our study demonstrates that the production of IL-6 increased significantly after treating with LPS within 24h.The levels of IL-6 and IL-1βfrom the right cortex microglia are higher than the left, because of the cytokines are associate with the brain lateralization, then we assume that are the LPS receptors in the microglia also asymmetric? The purpose of this report is to study the cytokines secreting ability between the right and left cortical microglia, analyze their roles in the relationship between the immune system and the brain lateralization. In present work, we aim to investigate the cytokines (IL-6 and IL-1β) levels secret from microglia that related to brain lateralization before and after LPS stimulation; and to explore the LPS receptors express in the microglia related to brain lateralization. Materials and MethodsCultures were prepared from the brains of 1-day-old BALB/c mice, the animals were killed by decapitation, whole brain was cleared from adhering meninges and blood vessels and mechanically dissociated, the left and the right cerebral cortices were separated and plated into different tubes. The fibroblasts were removed by allowing the cells to adhere to the surface of plate for 30 min, after turn up the plate, the cells which can not stick are glial cells. The primary right and left glial cell cultures were maintained in Dulbecco modified Eagle medium-Ham F12(1:1) supplemented with 10% fetal calf serum in humidified atomosphere of 5% CO2/95% air at 37℃, and the growth medium was replaced after 24h, then replaced every 3 days. Use the mixed glial cells between DIV15 and DIV30 for isolation of microglia, wash mixed glial cells for 1 min in DMEM-F12 to eliminate serum, incubate cells at 37℃20-30 min with 3 ml per flask of trypsin 0.25%:DMEM-F12 =1:3 until intact layer is detached,add 3 ml of DMEM-F12 with 10% FCS for trypsin inactivation, isolated microglial cells can be recovered by a 5-min incubation with trypsin 0.25% with vigorous pipetting and replated. Microglial cells were reseeded with complete medium at a density of 5×105 cells/ml in 6-well plates for 24 hour at 37℃.Removed the medium and washed three times with DMEM-F12, Lipopolysaccharide(LPS; Escherichia coli serotype O55:B5; Sigma Chemical Co)was added in a final concentration of 10μg/ml in both left and right cortical microglia for 24 hours in the DMEM-F12(1:1).After exposure to LPS, culture medium samples were collected and centrifuged, then stored at -80℃until analysis. The cytokine levels were determined by ELISA kits, and the cells in the 6-well plates were used for analyze the LPS receptors by RT-PCR. Results1. IL-6,IL-1βlevels of microglia in BALB/c mice:①In normal group, the microglia secret slight IL-6,IL-6 level in right microglia was higher than the left but had no statistic significance; when treated with LPS, both sides were elevated and the right was higher than the left(P<0.05).②For IL-1β, In normal group, the microglia secret slight IL-1β, and the level of IL-1βin right microglia was higher than the left(P<0.05); after stimulated by LPS, the IL-1βlevel was much higher than that of the normal group(P<0.05).2. LPS receptors expression in the brain of microglia in BALB/c mice In RAN level①By RT-PCR, CD14 expressed in microglia, and the level of CD14 in right side of microglia was higher than the left, after stimulated by LPS, the expression of CD14 was up-regulation.②By RT-PCR, TLR4 expressed in microglia, but MD-2 expressed slightly, and the right cortical microglia expressed more TLR4 than the left, when treated with LPS, the level of TLR4 was down regulation.Conclusion1. High-Yield isolation of murine microglia by mild trypsinization.2. The right microglia is found to be more sensitive to the elevation of IL-6 induced by LPS, the level of IL-6 released by microglia are higher in the right than those of in the left.3. There is a significant difference in IL-1βlevel which released by the right microglia stimulated by LPS, the level of IL-1βare higher in the right than that of in the left.4. There is a obvious difference in LPS receptors expression in microglia, the right microglia express more than the left, and after stimulated by LPS, the expression of CD14 was up-regulation, but the level of TLR4 was down regulation.
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