Effect Of Microglia Activation On Dopaminergic Neuron Injury Induced By Manganese And Its Possible Mechanism

As an essential trace element, manganese can balance the physiological function of human body, however, it can cause toxic injury with excessive intake. In the recent years, with the development of economy and the environmental change of industry, the content of environmental manganese is gradually on the rise. So people attach great importance to the damage by its toxicity. The main target organ of manganese is nervous system. At the early stage, its major symptoms are neurasthenic syndrome and autonomic nerve functional disorder, and then the apparent damage to extracorticospinal tract nerves, similar to that of a Parkinson's disease (PD). The epidemiologic study shows that manganese is an environmental risk factor to the incidence of PD.In the past, the study on the toxicity of manganese used to focus on the neuron injury especially direct dopaminergic neuron injury, while the effects of glial cells on the nerve toxicity by manganese were always neglected. Microglia cell, an important resident immunoreactive cells in the CNS, its activation is not only responsible for the cleavage of dead cell debris, but also involved in the initiation and propagation of the neuron injury. Excessive release of a host of factors released by activated microglia, such as nitric oxide (NO), tumor necrosis factor alpha (TNF-a) and interleukin 1β(IL-1β) was believed to be detrimental to neuronal survival. These proinflammatory or reactive oxidative species-induced cascade formed into a deleterious loop which finally lead to cell injury or death.In the present study, we try to explore the relationship between the activation of microglia and the neurotoxicity of manganese, to determine whether inhibition of microglia activation could protect the manganese induced dopaminergic neuron injury and to clarify the possible machenism of manganese induced neurotoxicity.Aim:1.To study the effect of manganese on microglia activation and dopaminergic neurons injury by using the Transwell model, co-culture microglia cells and dopaminergic neurons.2. The aim of the present study was to explore the possible mechanism of microglia activation in manganese induced dopaminergic neuron injury. Furthermore to study whether SMT and Minocycline could protect the dopaminergic neurons from toxicity induced by manganese and to clarify the possible mechanisms of the neuroprotection.Methods:1. A model of Transwell has been developed for the co-culture nearly pure microglia cells and dopaminergic neurons.2. Microglia activation was detected by the immunohistochemical analysis; Dopaminergic neurons injury was measured by immunostaining and TUNEL. 3. The level of iNOS was determined by immunostaining with iNOS antibody. The level of IL-1βand TNF-αwere detected by enzyme-linked immunosorbent assay.Results:1. The primary cultured technique described here allows culture of highly purified microglia and dopaminergic neurons by purification and identification with immunocytochemistry stain. The rate of purification are up to 98% and 2%, respectively. It is suitable for establishment of coculture model.2. Microglia were treated for 48h with 300μmol/L MnCl2, then were removed from the culture plate and immediately cocultured with dopaminergic neurons for 48h in Transwell plates. Microglial activation was determined by immunostaining. The results show that the rate of microglia activated by manganese treatment(75.1%±8.6%) is significantly higher than control(26.7%±4.5%), it revealed that manganese might induce activation of microglia. Immunocytochemical analysis for TH-positive neurons demonstrated that application of manganese induced a significant decrease in the number of dopaminergic neurons(6.8±0.73) compare to control(39.71±2.86). It is suggested that manganese can injury dopaminergic neurons.3. Purity of microglia were stimulated with 0μmol/L MnCl2, 300μmol/L MnCl2 or 300μmol/L MnCl2 combined 300μmol/L SMT for 48h .Then were removed from the culture plate and immediately cocultured with dopaminergic neurons for 48h in Transwell plate. The level of iNOS was measured using the immunocytochemical analysis. Significant induction of the manganese-induced iNOS protein was observed in cells compared with the control. SMT had inhibitory effects on manganese-induced production of iNOS. Immunocytochemical analysis for TH-positive neurons demonstrated that treatment with SMT could effectively reduce the decrease in the number of dopaminergic neurons.4. Purity of microglia were stimulated with 0μmol/L MnCl2, 300μmol/L MnCl2 or 300μmol/L MnCl2 combined 45μmol/L Minocycline for 48h.Then were removed from the culture plate and immediately cocultured with dopaminergic neurons for 48h in Transwell plate. The results showed that Minocycline decreased the number of manganese-induced microglial activation. Further, Minocycline significantly reduced the release of activated products (IL-1β; TNF-a; iNOS) after manganese-induced microglial activation and attenuated manganese-induced the decreased the number of dopaminergic neurons.Conclusions:1. The model of cocultured of primary microglia and dopaminergic neuron in Transwell plate was well established in our experiment. It is feasible and creditable for the further study of the role of microglia activation on dopaminergic neuron injury induced by manganese and its possible mechanism.2. It was confirmed that micrglia activation and dopaminergic neuron injury could be induced by moderate manganese.3. The results indicated that microglia activation and up-regulation activated products might be involved in the mechanism of manganese induced dopaminergic neuron injury. Minocycline and SMT protected the dopaminergic neuron injury induced by manganese might be through inhibiting microglia activation and the expression of activated products respectively.