Effect Of Activated Microglia On SOD Activity And MDA Content In Cortical Neurons

Objective: Diffuse axonal injury (DAI) is seen as wide-spread damagecharacterized by neurons neuraxial swelling,fracture, which can occur itselfor concomitantly with other brain injury. It is one of the most common causesto cause head injury patients died, heavy remnants and plants survivalstate.The model performance were disorder of consciousness and theprognosis was poor. Recent studies have shown that the functional disturbanceinduced by DAI was evoked by the shear or tensile forces generated by theprimary injury, but also caused by the "secondary attack", or neuronsecondary injury,happened in the following time after the injury. Thesecondary injury refers to the delay neuraxial fracture caused by a series ofbiochemical and cells reaction after injury.Our previous result showed that a large number of Microglia cells wereactivated around the beta APP positive expression of neuraxial. Some researchresults have showed that activated microglia could create considerable freeradicles which could cause lipid peroxidation and injury neuron. SOD whichexist in the cytoplasm and mitochondria is a metal protease, its performancecan be indirectly reflect the content of free radicals in organization and thedegree of lipid peroxidation. MDA which product by lipid peroxidation afterfree radicals attack biological membrane is a lipid peroxide, the contentchange can be directly reflect the severity of the attack by free radicals to thecells. Therefore, the mechanism of nerve damage not only limited to directeffect of cytokine, the toxicity of activated microglia are more importantly.However, whether microglial cells can be through the way of oxidativedamage lead to cortical neurons late-occurred damage in vitro cultureconditions have not been reported.Our study was designing to give a certain amount of Aβ1-40to inductionmicroglia, and then stimulate cortical neurons with its conditions medium in vitro culture conditions. We can observation whether the microglia couldcause oxidative damage to cortical neurons through the index changes of SOD,MDA, and discussed the interaction between activated microglia and corticalneurons in vitro experiments.Methods: Microglial cells in cortex from new SD rat (within24h) werecultured in vitro. Induction activated microglia by different concentration ofsoluble Aβ1-40, and then determine the best state of activated microglia throughthe expression of TNFα and IL-1β was detected by ELISA．The expression ofTNFα and IL-1β was detected by ELISA and Real Time PCR after joinminocycline. And then stimulated cortical neurons by activated microgliacondition mediums and divided into four groups randomly: control,1/2Aβ-MCM group,1/4Aβ-MCM group,1/8Aβ-MCM group. each groupinclude five phase:1h,3h,6h,12h and24h.The activity of superoxidedismutase(SOD)was measured with xanthine oxidase method, the content ofmalondialdehyde(MDA)was detected with barbituric acid reactionchromometry, this will serve to explore the degree of oxidation injury of brainneurons.The data were presented as Mean±SD, analyzed with ANOVA and LSDusing SPSS13.0statistical program. A level of p<0.05was considered asstatistical significance.Results:1Induction microglia cells activated by Aβ:ELISA results showed that1μM Aβ1-40incubation microglia cells6h can succeed induction microglia cellsactivated to secretion TNFα, IL-1β, significantly higher than those of thecontrolled group (p<0.01), and the induction activation function have time andconcentration dependence. Real Time PCR and ELISA results indicate thatminocycline can significantly inhibited TNFα and IL-1β secretion of microgliaafter add0.2μM minocycline, MC+Aβ group dropped significantly comparedwith Aβ group (p<0.01), and has no significant difference compared with thecontrol group(p>0.05); However0.2μM minocycline alone have no effect onthe content of TNFα and IL-1β. So, we choose final concentration of1μM Aβ1-40to incubation microglial cells for6h, then the supernatant as the bestcondition mediums was added to cultured cortical neurons.2The oxidatie stress damage of cortical neurons by Microglia conditionmediums:The results indicated that SOD was significantly lower in corticalneurons than that in the controuls,which stimulated by conditions medium ofdifferent concentration,1/2Aβ-MCM group was the most obvious,24h pointwas the minimum, have obviously statistical significance compared with thecontrol (P<0.05), and had low tendency with time prolonged and dose raised.Added1/4or1/8Aβ-MCM to neuronal culture medium, the expression ofSOD showed no significant difference compared with control group.And thecontent of MDA were higher than those in the control group,1/2Aβ-MCMgroup was the most obvious,24h point was the peak, have obviously statisticalsignificance compared with the control (P<0.05), and had ascendent tendencywith time prolonged and dose raised.Conclusions:1Established an cortical neurons oxidatie stress damage model by theactivated microglia conditioned medium.2Activated microglia cells may caused the cortical neurons damagethrough the oxidative stress.