A Study Of The Toxic Effect And It's Mechanism Of Proteasomal Inhibitor On Cultured Midbrain Slices

Part one Establish the model of midbrain slices in vitro cultureObjective: To establish the long-term model of organotypic midbrain slices in vitro culture in order to build up a system for the further studies of drug interventions. Methods:Take the method of liquid-atmosphere interfacial culture system, 10-28 days old Wistar rat's midbrain slices were cultured on the Millicell-culture insets membranes for 10-30 days. The vitality of the slices were identified by measurement of lactate dehydrogenase (LDH) released into the medium from the slices, the status of nigral neurons were investigated by HE staining and TH(tyrosine hydroxylase) immunohistochemistry. Results:The brain slices can well grew in 10-20 days but gradually died after that. LDH detection shows that the midbrain slices were stably cultured in 5-7 days. The anatomic structure of the slices and nigra neurons were well present on HE and TH staining. Conclusions: The cultured brain slice can well grow in 7-21 days, the long-term model of organotypic midbrain slices in vitro culture was established successfully. Part two A study of the toxic effect and it's mechanism of proteasomal inhibitor on cultured midbrain slicesObjectiv:To observe the toxic effect of proteasomal inhibitor on dopaminergic neurons in cultured slices of the midbrain and to explore its roles. Methods:A long-term midbrain slice culture system of Wistar rats was established, after proteasomal inhibitor, lactacystin(0.1μmol/L,0.5μmol/L,1.0μmol/L,5.0μmol/L), were added into the culture system for 24h, the vitality of the slices were identified by measurement of lactate dehydrogenase (LDH) released into the medium from the slices. The loss and degeneration of nigral neurons were investigated by tyrosine hydroxylase immunohistochemistry,α-synuclein expression was observed byα-synuclein immunohistochemistry. The apoptosis of dopaminergic neurons was examined by TUNEL stain. Results:After treatment with proteasomal inhibitor lactacystin for 24 hours, the numbers of tyrosine hydroxylase positive cells in substantia nigra were decreased and the expression ofα-synuclein was increased(P<0.05). The apoptotic analyses by TUNEL showed that positive neurons were increased significantly in 0.5μmol/L,1.0μmol/L and 5.0μmol/L lactacystin group(P<0.05). Conclusions: Proteasomal inhibitor lactacystin has toxic effect to dopaminergic neurons on midbrain slices and induce apoptosis of nigral neurons. The underlying mechanism may be due to the accumulation ofα-synuclein. Proteasome dysfunction may play an important role in the pathogenesis of Parkinson's disease.