| Background and SignificanceOngoing chronic inflammatory process in the brain may underlie the pathologies of several neurological disorders, including Parkinson's disease and Alzheimer's disease. Microglia cells, the resident immunoreactive cells in the brain, lies in the core of the this process. Microglial activation is not merely responsible for the cleavage of died cell debris, but also actively involved in the initiation and propagation of the neuronal cell 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 ip (IL-1B) are 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. Based on finding that microglia activation plays a pivotal role in the pathogenesis of Parkinson's disease, a newly conception that inhibition of microglial activation may be a novel target to dampen the progress of the disease is emerging. The illustration of mechanisms for microglial activation and the establishment of drug-screen system are largely depending on the abundant highly purified microglia and the matching cell model. As a major component ofgreen tea polyphenol, (-)-epigallocatechin gallate (EGCG) has been intensively investigated in regard to its excellent anti-oxidative effect and anti-inflammatory action in peripheral system. If EGCG possesses the property to inhibit microglial activation, which is profoundly involved in the neurodegenerative diseases in the central nervous system, is still to be illustrated.Specific aim1. To establish a system with which a relatively large scale drug-testing related to microglial activation-induced neuronal injury can be fulfilled.2. To test the reliability and feasibility of the developed system.3. To explore the effect of a monomer of green tea polyphenol, (-)-epigallocatechin gallate (EGCG), on microglia activation in vivo.4. To investigate EGCG's action on microglial activation and the underlying mechanism.Methods1. We treated primary mesencephalic cell cultures with neurotoxin methyl-4-phenyl-pyridinium (MPP+) to induce dopaminergic neuron injury and tested the effects of EGCG at different doses on dopaminergic neuronal survival through the examination of tyrosine hydroxylase-immunoreactive (TH-ir) cells in the cultures.2. With unbiased stereological method in vivo, microglial activation and TH-ir positive cells were observed with specific cell marker CD11b and TH immunostaining method in A8, A9 and A10 region of substantia nigra (SN) in 1-methyl-4-phenyl-1, 2, 3, 6 - tetrahydropyridine (MPTP)-lesioned C57BL/6 mice, an standard animal model of Parkinson's disease.3. We used two reported protocols, namely, shaking method and mild trypsinization method, to harvested microglia, and compared their virtues inregard to their yield ability, morphological state and the profile of the microglia secretion of the two key factors, nitric oxide (NO) and tumor necrosis factor alpha (TNF- a ), which is the potent promoter of inflammatory response.4. Further, we observed the effects of the LPS-activated microglia culture conditioned medium on the viability of all-trans retinoic acid-differentiated human neuroblastoma cell line SH-SY5Y cell using MTT assay and 3H-dopamine uptake.5. In highly purified microglia culture, using DiI-ac-LDL labeling, we tested effects of EGCG on lipopolysaccharide(LPS)-induced morphological change of microglia activation. We also investigated the effects of EGCG upon NO and TNF-a release by activated microglia.6. More over, Western blot analysis and semi-quantitive RT-PCR were used to determine weather the inhibitive effect of EGCG on microglial released NO and TNF-a was the consequences of iNOS and TNF-a mRNA downregulation in cytoplasm of microglia.7. We utilized purified microglial culture combined with...
|
PDF Full Text Request