| Parkinson's disease (PD) is the second most common neurodegenerative disease. It's typical symptom includes resting tremor bradykinesia and other movement disorders.A lot of patients may be associated with mood disorders, cognitive impairment or dementia and other non-motor symptoms. However, the exact causes of PD and mechanisms remain unclear. Epidemiological survey of PD has revealed that the environmental factors are associated with the development of PD.Pesticide residual is an important source of environmental chemical pollutants. In China, the problem of pesticide residues exceeding exists for a long time. Recently many media reported about the "poisonous bean" with11pesticide residues exceeding the maximum allowed limits were never monitored. In the United States, organophosphate pesticide chlorpyrifos (CPF) and it's metabolites were detected in the urine of almost82%of the surveyed population. Chlorpyrifos is widely used all over the world. It is widely used for family life, agricultural production and public health programs as an insecticide and herbicide. The study of the chlorpyrifos'effects on dopaminergic neurons in the mesencephalic substantia nigra of rats thus may reveal the pathogenesis of PD and provide insights for the disease. This study will be explored from the following five parts.The first part:The effects of low-dose chlorpyrifos on the dopaminergic SH-SY5Y cellObjective:The study is designed to explore the effects of low-dose chlorpyrifos on dopaminergic SH-SY5Y cells. Method:Undifferentiated dopaminergic SH-SY5Y cells are exposed to chlorpyrifos (0uM,10uM,30uM,50uM and80uM)at the logarithmic growth phase, each of these groups was treated for different time(24h,48h,72h and96h).Cell morphology was monitored with inverse microscopy, The cell viability was calculated by using the MTT assay.Flow cytometry was used to quantitatively detect the apoptosis percentage of cells after exposured to chlorpyrifos for96h. Result:No morphological changes and apparent apoptosis were observed after the CPF induction. However, MTT assay showed that the SH-SY5Y growth was significantly inhibited (p<0.05)96h after the CPF (30uM,50uM,80uM) treatment. Conclusion: Exposure to low-dose(≤80uM) chlorpyrifos will not lead to apoptosis and morphological changes of dopaminergic SH-SY5Y cells, but CPF could affect the cell proliferation and viability at and beyond the dosage of30uM.The second part:The rat model for sub-threshold dosage chlorpyrifos exposure at the age of brain developmentObjective:The study is designed to establish a rat model of sub-threshold dosage chlorpyrifos exposure at the age of brain development. Method:Postnatal11days SD rats were randomly divided in to CPF group and control groups, and control groups then further divided into the dimethysulfoxide (DMSO) group and saline (NS)group. Postnatal11-14days rats were injected with CPF at sub-threshold dosage (i.c.5mg/kg/day in1ml DMSO). The control groups were injected the same dosage of DMSO or saline respectively. The acute symptoms of organophosphorus pesticide intoxicates, right reflex and twitch were checked12hours later after the dosage.The animals were sacrificed at three differernt time points (i.c. PND20, PND30, PND60) to examine the body weight gain, the pathology of the brain tissue, the coefficient of the brain and the water content of brain tissue. Results:SD rats in every group did not show any symptoms of acute organophosphate pesticide poisoning. The absolute value of body weight gain of each experiment group and the rate of body weight gain were not significantly different from those of the control groups (p>0.05).The outward appearance of brain tissue of every group was normal. The coefficient of the brain and the water content of brain tissue of experiment group were not significantly different from that of control groups (p>0.05). Conclusion: The rat model of sub-threshold dosage chlorpyrifos exposure was established at the age of brain development, sub-threshold dosage of chlorpyrifos exposure is easy to be considered as "safe", and thus is easy to be ignored.The third part:The effects of sub-threshold dosage of chlorpyrifos on the astrocytes, microglia and dopaminergic neurons in the mesencephalic substantia nigra of rats at the age of brain developmentObjective:The study is designed to explore the effects of sub-threshold dosage of chlorpyrifos on the astrocytes, microglia and dopaminergic neurons in the mesencephalic substantia nigra of rats when exposed in the brain developmental period. Method:Establish a rat model of sub-threshold dosage chlorpyrifos exposure to rats when they were in the period of brain development. The animals were randomly divided in to CPF group, DMSO group and NS group. The animals were sacrificed at PND20, PND30, PND60to examine the effects on midbrain dopaminergic neurons by immunohistochemical straining and immune fluorescent double labeling for tyrosine hydroxylase (TH) expression. More animals were sacrificed at12h,24h, and72h after chlorpyrifos exposure and at the time points of PND30, PND60. Meanwhile,the astrocytes and microglial cells were checked for glial fibrillary acidic protein (GFAP) and microglial surface marker (CD11b) through immunohistochemical straining and immune fluorescent double labeling. Transmission electronic microscope was used to examine ultrastructural changes of DA neurons when the rats were60-days-old. Results:CPF induced microglial and astrocytes activation in the substantia nigra, but CPF exposure decreased the level of TH immunoreactivity in the substantia nigra of rats since30-days-old. Some of the DA neurons' ultrastructures in the substantia nigra were changed. Conclusion:The sub-threshold dosage chlorpyrifos exposure to rats could induce rapid activation of microglia and astrocytes in the substantia nigra at the age of brain development. Such exposure may even lead to dopaminergic neurons loss in the substantia nigra at the later life stage.The fourth part:The effects of sub-threshold dosage of chlorpyrifos exposed to developmental brain of rats on the p38MAPK, NF κ B p65signaling pathwaysObjective:To investigate the effects of sub-threshold dosage of chlorpyrifos on the important inflammatory signaling pathways----p38MAPK and NF κ B p65. Method:Establish a rat model of sub-threshold dosage chlorpyrifos exposure to rats when they were in the period of brain development. The animals were randomly divided in to CPF group,DMSO group and NS group. The animals were sacrificed at12h,24h, and72h after chlorpyrifos exposure. The effects on p38MAPK and NF κ Bp65signaling pathways were examined by both immunohistochemical straining and western-blotting.Results:The p38MAPK signal pathway was activated rapidly after chlorpyrifos exposure. As for the NF κ B p65pathway, the NF κ B p65nuclear staining positive cells were increased after chlorpyrifos exposure. The total amount of p-NF κ B p65in the substantia nigra was increased significantly, However, the amount of NF κ B p65remained the same after the chlorpyrifos exposure. Conclusion:Sub-threshold dosage of chlorpyrifos exposed to developmental brain of rats could quickly active inflammatory signaling pathways,such as p38MAPK and NF κ B p65signaling pathway.Such activation may suggest dopaminergic neurons damage in the later life stage.The fifth part:The sub-threshold dosage of chlorpyrifos exposed to the developmental brain of rats changed the neural behavior at their adolescence and adulthoodObjective:To investigate the effects of sub-threshold dosage of chlorpyrifos exposure on the neural behavior of rats in the later life stage. Method:Established a rat model of sub-threshold dosage chlorpyrifos exposure at the age of brain development. The animals were randomly divided in to CPF group, DMSO group and NS group. Then animals were fed until they were PND30or PND60old. Then open field test, grip strength test, the slope test and the Morris water maze test were used to examine the neurobehavioral changes in rats. Results:The total number of lattice in the open field test of CPF group rats was less than that of control groups at the age of PND30; but the rearing number and modification frequency of the CPF group were less than those of the control groups' at the age of PND60;and the stool numbers of CPF group were bigger than those of the control groups at the age of PND60. The total time of CPF group rats to catch the rope in the grip traction test is less than the control groups'at the age of PND60and the rats of the CPF group were easier to slide down in the slopes experiment. The escape latency of the CPF group was more than that of the control groups'and the swimming distance percentage in the object quadrant of the CPF group was less than that of the control groups'in the Mirros water maze test at the age of PND60. Conclusion:Decreased motor activity and body coordination ability were observed in the CPF group at the age of PND30and apparent memory impairment was observed of the CPF group rats when they were60-days-old.
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