Murine Microglial Cells With M2Subtype Prevent Hippocampus Neurons From Neurotoxicity Induced By Kainic Acid

Excitotoxicity is one of the important pathogenesis of variousneurodegenerative diseases of human CNS. Excitotoxic injury induced byKainic acid (KA) is an effective model to study neurodegenerativediseases. Some studies have confirmed that activations of microglia andastrocytes were involved in the process of KA induced neuronal death.The function of microglia has two sides: M1subtype can performpro-inflammatory function and M2act as anti-inflammatoryone. Therefore, this experiment will use different cytokines to inducemicroglial cells differentiating into different subtypes, then clarify theprotective role of M2subtype against KA-induced hippocampusneurons damage and its ant-inflammatory mechanism. We suppose toprovide a theoretical basis for the clinical treatment of neurodegenerativediseases.Objective:The study use primary hippocampus neurons neurotoxic injuryinduced by KA to establish neurodegenerative model. We also do primarymicroglial cells culture from neonatal mice, then seperate anddifferentiate the microglial cells. Then build up the co-culture model ofinduced microglial cells and hippocampal neurons in vitro, to investigatethe different role of different subtypes of microglia in the process ofneurotoxicityinduced by KA. We supposed to clarify that M2subtypemicroglia could prevent hippocampal neurons damage from neurotoxicityinduced by KA and its anti-inflammatory mechanism. Method:1）Use E16-18fetal mice to do primary hippocampal neurons culture,then establish neuronal excitotoxic injury model with KA treatment,evaluate the degree and mechanism of neuronal damage with CCK8, NOand LDH production;2) Use P1-3neonatal mice to do primary microglialcells culture, then sort microglia with magnetic beads and stimulate withcytokines to differentiate. Cellular pheotype were analyzed by flowcytometry to identify the markers of induced microglia cells (M1/M2).Use ELISA to determinate the cytokines secreted by different subtypes;3)Build up the co-culture model of hippocampal neurons and inducedmicroglial cells in vitro, then test CCK8, NO, LDH to evaluate thedamage of neurons after co-culture. After add KA treatment for24hours,observe different regulatory effects of M1/M2on KA-inducedneurotoxicity by CCK8and LDH test, then study to expressional level ofNFκB by Western Blot tests.Results:1) KA treatment could provide a well-characterized model ofneurodegenerative diseases. After24h for200uM KA treatment, most ofthe neurons were dead with axon damage. Our results showed that KAtreatment reduced neuron survival to51.7±8.6%, while NO and LDHproduction in hippocampus neuron supernatants increased apparentlyafter KA treatment. Western Blot results suggest that expressions ofNFκB and Caspase3were significantly increased after KA treatment.2) Primary glial cells from neonatal mice were cultured and thenseparated using anti-CD11b MicroBeads. The positive rate of CD11b was92.7±5.3%.3) M1phenotype of microglia cells were successfully induced bycombined stimulation of LPS and IFN-γ, showing "amoeboid" activation, with larger cellular body and retracted neuritis. FACS test showed thatthe phenotype of M1was iNOS+，CD40+， IL-12/23p40high，IL-10low.ELISA results showed that the induced-M1microglia could secrete highlevels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6), significantlyhigher than that in control group and M2(P<0.01, n=3).4）M2phenotype of microglia cells were successfully induced byIL-4, IL-10, and TGF-β combined stimulation, showing smaller cells withless longer processes. FACS test showed that the phenotype of M2wasArg-1+，CD206+，IL-10high，IL-12/23p40low. ELISA results showed thatthe induced-M2microglia could secrete high levels of anti-inflammatorycytokines (IL-4and IL-10), significantly higher than that in control groupand M1(P<0.01, n=3).5) Successfully establish co-culture model of hippocampus neuronsand induced-microglial cells in vitro. M1microglia could cause apparentneuron death and axonal fracture after co-culture, the activity of CCK8was51.59%±19.40%(n=3) with higher production of NO. Whereasthere is no significant changes after M2co-culturing, CCK8was96.60%±3.21%(n=3). Western Blot tests showed that the expression of NFκBin N+M1group was significantly higher than N+M2group, there wassignificant difference between two groups (P<0.01).6) After200uM KA treatment for24h, most hippocampus neuronswith M1cells co-cultured were dead with axon damages, neuronalactivity in CCK8was32.36%±11.13%, NO yield increased significantly.While neurons co-cultured with M2microglia acted on the contrary,CCK8was80.39%±11.81%. The expression of NFκB in N+M1+KAgroup were significantly higher than that in N+M2+KA group, there wassignificant difference between two groups (P<0.05). Conclusions:(1) KA treatment could provide a well-characterized model ofneuronal excitotoxic damage in vitro. KA may play a role in inducing theapoptosis of neurons through the up regulation of NFκB and Caspase3signal pathway.(2) M1phenotype of microglia cells were successfully induced bycombined stimulation of LPS and IFN-γ, M2phenotype of microglia cellswere successfully induced by IL-4, IL-10, and TGF-β. Differentiationalmicrogllial cells could express the characterized phenotype markersand secret corresponding cytokines.(3) Induced M1microglia can cause the damage of co-culturedhippocampal neurons and worsen the damage of neurons caused by KA,whereas induced-M2subtype has no effect on co-cultured hippocampalneurons and M2could prevent the hippocampus neurons fromneurotoxicity induced by KA.(4) M2subtype microglia may protect neurons from KA throughsecreting of anti-inflammatory cytokines, inhibiting production of NOand down regulation of NFκB signaling pathways.