Promising Neuroprotective Function For M2 Microglia In Kainic Acid-Induced C57BL/6 Mice Neurotoxicity Via The Down-Regulation Of NF-?B And Caspase 3signaling Pathways In Vitro

Objective:Neuroexcitotoxicity is the most important reason of neural loss of neurodegenerative diseases.Kainic acid,an analogue of glutamate,which induced neuroexcitotoxicity is considered as the classic neurodegerative disease model in vivo and in viro.Microglial cells are resident imuunne cells in central nervous system,play very important roles on immunal survey,immunal regulation and circumstance homeostasis.Microglia can be activated as two functional states(M1 and M2)which play dual roles in neurodegenerative diseases.M1 phenotype,the traditional classical phenotype,which is polarized and aggregated at the progressing stage of neurodegenerative disease,secrets amount of proinflammatory cytokines,acts proinflammtaory roles in central nervous system;M2 phenotype,the altered phenotype,which is polarized at the primary and recovery pahse of neurodegenerative disease,phagocyts pathogens and secrets abundant of anti-inflammatoy cytokines,and promotes the tissue repair,plays a neuroprotectvie role on damaged neurons.Changing the phenotype of M1 toward to M2 phenotype may diverse the prognosis of neurodegenerative diseases.In this present study,we explored a possible neuroprotective function mechanism of M2 microglia against kainic acid(KA)-induced neurodegenerationMethods:Neurons were isolated from the hippocampi and cortices of C57BL/6 embryos(embryonic day 16)and cortical microglia were extracted from neonatal pups(postnatal days 0-2),nerons and microglia were primarily cultured respectively.Microglia were classified as three phenotypes MO-phenotype(unstimulated/primarily cultured microglia);stimulated with lipopoly saccharide and interferon-y to form the M1-phenotype;stimulated with interleukin(IL)-4,IL-10,and transforming growth factor-? for the M2-phenotype.Next,we separated the different phenotype of microglia and tested the markers respectively by cytomery technology.Neurons were co-cultured with each of the three microglial phenotypes and treated with KA for 24 h.Finally,we analyzed the cell survival rate,nitric oxide(NO)levels,and lactate dehydrogenase production,cytokines levels,and expression of nuclear factor ?B(NF-?B)and caspase 3 among the four groups(N,N+MO,N+M1,N+M2)before and after KA insult.Data were presented as mean value±standard deviation(SD),and differences were evaluated by student's t-test or analysis of variance.The paired student's t-test was used to analyze the data of neuron culture alone,one factor analysis of variance(one-way ANOVA)followed Tukey's test were used to analyze the results from cytometry,and two factors analysis of variance(two-way ANOVA)followed Benferroni posttests were used to analyze the data of neuron co cultures with each phenotype of microglia respectivelyResults:Our results indicated that(1)Neurons damaged apparently after KA insult:the morphology of neuronal cells changed apparently after KA stimulation,neuronal survival decreased significantly while production of NO and LDH incressed significantly(P<0.05,n=3);(2)M1 microglia enhanced the KA-induced neurotoxicity while M2 microglia played a neuroprotective role in KA-induced neurotoxicity:? After KA stimulation,neuronal survival of neurons co-cultured with M1 microglia(N+M1)comapred with neurons cultured anlone(N)was decreased significantly(P<0.05,n=3),while neuronal survival of neurons co-cultured with M2 microglia(N+M2)increased significantly compared to neurons cultured anlone(N)after KA insult(P<0.05,n=3);?After KA treated,neurons co-cultured with M1 microglia(N+M1)exhibited higher production of NO than neurons co-cultured with MO microglia(N+MO)or neurons co-cultured with M2 microglia(N+M2)did(P<0.001,n=3);?The level of pro-inflammatory cytokines TNF-? and IL-6 from supernacants of neurons co-cultured with M1 microglia(N+M1)were higher than those from supernacants of neurons co-cultured with MO microglia(N+MO)or neurons co-cultured with M2 microglia(N+M2)after KA stimulation(P<0.001,n=3);?The level of IFN-y from neurons co-cultured with M2 microglia(N+M2)of after KA insult was lower than that of group of before KA insult(P<0.01,n=3);(3)The expression of NF-?B and caspase 3 were significantly decreased in M2 microglia co-cultures compared to M1 or MO microglia co-cultures after KA insult.Conclusion:?KA-induced neuroexcitotoxiticiy is a stable neurodegen-erative model;?M1 microglia enhanced the KA-incuded neurotoxicity while M2 microglia played a neuroprotective role in KA-induced neurotoxicity;?M2 microglia may exert a neuroprotective function in KA-induced neurotoxicity via the down-regulation of NF-?B and caspase 3 signaling pathways.