Exploration Apoptosis Pathway And Necroptosis Of Nerve Cells In PD Rat Model

Parkinson disease (PD) is a neurodegeneration disease that characterized by aprogressive loss of dopaminergic neurons in substantia nigra and depletion of theneurotransmitter dopamine in striatum.It is difficult to explain the phenomenon thatneurons loss extremely slow and characteristic pathological changes of the remnants bycurrently classical apoptosis theory.Objective：To observe the way of cell death(apoptosis and necroptosis) ofdopaminergic neuronal lesions in SN and STR of rat, expression and significance of keyproteins of apoptosis pathway. To research neuroprotection of Caspase inhibitor(Z-VAD-fmk) on the6-OHDA rat model of PD damage and effects on nerve cell death,to provide the scientific theoretical evidence for the pathogenesis of neurodegenerativediseases and clinical prevention.Methods:(1) The models of PD were established by injecting6-OHDA in theright medial forebrain bundle(MFB) of rats, others equivalent saline and served ascontrols. To observe the loss of neurons in the striatum and substantia nigra(SN) of PDrat, HE staining and Nissl staining was performed.(2) The change of expression ofTNF-α、P53、P21、Bcl-2/Bax、Casepase-3were examined by immunohistochemistry.(3) To realize of repeated Z-VAD-fmk injection, improved the methods of the rat braincannulation administration. Z-VAD-fmk were injected into the PD rat MFB through theself-made cannula, equivalent5%DMSO as PD and control group.(4) Nissl staining wasused to observe the loss of neurons and in PD after Z-VAD-fmk injection.Ultrastructurechanges were observed by Transmission electron microscopy；(5) Bcl-2,Bax,RIP1,RIP3mRNA were detected by real-time PCR.Results:(1) The typical ipsillateral rotations over210in30minutes were occurredin the second week.90%neurons of the right substantia nigra compacta(SNc) inlesioned side of the rat model of PD were not seen in Nissl dyeing and THimmunofluorescence staining.(2) Immunohistochemical analysis demonstrated that exprsssinon of TNF-α、P53、P31、Bax、Casepase-3were upregulated, Bcl-2wasdownregulatd.(3) Z-VAD-fmk improved rotation behavior and neuron loss as well asupregulated Bcl-2/Bax mRNA in PD rats.(4) Z-VAD-fmk improved the ultrastructurechanges in PD rat, meanwhile, part of the glial cells appear edema.(5)RIP1, RIP3mRNA upregulated（P<0.05）,which indicate that neurons by enabling selectivenecroptosis of glial cells.Conclusions:(1)Apoptosis is the main way of cell dead in PD rat model.(2) AfterZ-VAD-fmk injection, to some extent,programmed cell death was inhibited, glial cellsgudergo necroptosis.(3) Z-VAD-fmk protect neurons by enabling selective necroptosisof actived glial cells.