Role Of Microglia In Manganese-induced Dopaminergic Neuron Injury In Rat Substantia Nigra

The trace element manganese is essential for normal development and body functions across the life span of all mammals. But excess brain manganese produces neurotoxicity. Chronic manganism can cause some extracorticospinal tract symptoms, which is similar to Parkinson's disease. The pathological process of Parkinson's disease is selective degeneration of dopaminergic neuron in substantia striatal system, which leads to dopamine deficiency. But the exact mechanisms of manganese neurotoxicity are still being unraveled.In the past, the study on the manganese neurotoxicity used to focus on the neuron injury, especially the direct dopaminergic neuron injury caused by manganese, while the effects of glial cells on the manganese-induced neurotoxicity were always neglected. Microglia, the standby cells for immune defense in the central nervous system, monitor the brain environment. The most characteristic feature of microglial cells is their rapid activation in response to even minor pathological changes in the central nervous system. In the activated state, microglia can serve diverse beneficial functions essential to neuron survival. However, under other circumstances, microglia become overactivated and can induce significant and highly detrimental neurotoxic effects by the excess production of a large array of factors such as iNOS, TNFαand IL-1β.Recently evidences showed that manganese enhances proinflammatory factors and iNOS production by LPS-activated N9 or BV2 microglial cells. In the present study, we employed animal model to explore the role of microglia in manganese-induced dopaminergic neuron injury and the underlying mechanism in vivo, and offer some evidences for the elucidation of the mechanisms of manganese neurotoxicity.Objective:To establish the animal model of manganism by stereotaxic surgery, and to determine whether manganese can alter the activation state of microglial cells and induce the dopaminergic neuron injury in rat substantia nigra. To investigate the role of microglia in manganese-induced dopaminergic neuron injury and the underlying mechanism in this process.Method:①The animal model of manganism was established by stereotaxic injection into the rat striatum.②The density of the dopaminergic neurons was detected by immunohistochemical staining, and the activation state of microglial cells was detected by immunofluorescence staining.③The gene expression of iNOS and TNFαwas assayed by RT-PCR. The expression of iNOS was detected by immunohistochemical staining. The content of TNFαand IL-1βwas assayed by ELISA.Results :①We created the animal model of manganism by intrastriatal microinjections.②Resting microglial cells are highly branches with small cell bodies. In the activation state, the length of branches of microglial cells are reduced, and the cell body become larger. Our results showed that following 1 day of manganese administration, a few microglial cells became activated. 7 days after manganese, the number of activated microglial cells in the substantia nigra increased. In control group animals, no activated microglia was present.③The results of immunohistochemical staining showed that 1 day after manganese administration, no detectable difference in TH-ir was observed in the substantia nigra between the control group and the manganese-treated group. 7 days after Mn, there was a marked loss of TH-ir neurons in the manganese-treated group compared with the control group.④The gene expression measured by RT-PCR indicated that manganese upregulated the TNFαand iNOS mRNA in the striatum and substantia nigra. The protein expression of IL-1β,TNFαand iNOS detected by immunohistochemical staining and ELISA also increased compared with the control group.⑤Minocycline treatment attenuated microglial activation, mitigated both the formation of IL-1β,TNFαand iNOS and the dopaminergic neurotoxicity produced by manganese.Conclusion:Our research suggested that manganese can result in the selective dopaminergic neuron losses and the activation of microglial cells, and the activation product might play an important role in this process. We showed that minocycline that inhibits microglial activation independently of its antimicrobial properties, mitigated the damage of dopaminergic neuron produced by mananese. These findings offer further insight into the potential mechanisms of manganese neurotoxicity.