A Study Of Neurotoxicity Of Rotenone On Dopaminergic Neurons In The Cultured Slices Of The Substantia Nigra Of Rats

Part one An establishment the PD model with oganotypic slices of midbrain cultureObjective: To establish the long-term model of organotypic midbrain slices in vitro culture and make preparations for the further studies of Parkinson's disease. Methods:Take the midbrain slices from 10 days old wistar postnatal rat,cultured in the Millicell-culture insets membranes,and then were treated with low concentrations of rotenone.The morphology of the slices were observed through inverted microscope. The vitality of the slice was identified by the test of lactate dehydrogenase(LDH) release, The apoptotic analysis of dapaminergic neurons by study of TH(tyrosine hydroxylase) immunohistochemistry. Results:The brain slices can well grow in more than 20 days but gradually died after that. LDH detection show that the brain slices were stably cultured in 5-7 days. The anatomic structure of the slices and dopaminergic neurons were well present on TH staining.Conclusions: The cultured brain slice can well grew in more than 20 days, the long-term model of organotypic midbrain slices in vitro culture were established successfully. Part two A Study of neurotoxicity of rotenone on dopaminergic neurons in the cultured slices of the substantia nigra of ratsObjective:To investigate the early neurotoxicity of rotenone on dopaminergic neurons and explore an ideal tissue model and the protective effect of Edaravone. Methods: A long—term midbrain slice culture system of Wistar rats was established according to the interface tissue culture method.After rotenone was added for some time,its toxic effects on the whole slices and the dopaminergic neurons were identified through the measurements of LDH,GSH-Px,SOD,MDA released into the medium from the cultured slices, as well as the observations of immunohistochemistry for tyrosine hydroxylase(TH), detect the levels ofα-synuclein through western-blot; detect ultrastructure though electron microscope. Results: In those cultures exposed to rotenone for more than 20 days, the TH-positive neurons in tissue dramatically decreased with the concentrations rising, rotenone also inducedα-synuclein aggregation, edaravone has protective effect of these. Conclusion: Long-time stable midbrain slice culture system has been set up successfully. The neurotoxicity of rotenone on the whole slices and dopaminergic neurons shows a dose and time dependent manner.Rotenone inducesα-synuclein aggregation and oxidative damage in organotypic midbrain slice cultures, edaravone protects slices from rotenone-induced oxidative damage.