Neuroprotective Effect And Mechanism Of Melatonin On Mptp Mouse Model Of Parkinson’s Disease

Section 1 Impairments of behavior and pathology in subacute MPTP mouse model of PDObjective:To investigate stable behavior tests for the subacute MPTP mouse model of Parkinson’s disease(PD),find further verifications by method of pathology.Methods:C57BL/6 mice were randomly divided into two groups:NS group and MPTP group.Mice were given intraperitoneal injection of MPTP,at 30 mg/kg once a day for 5 days.Open-field,pole and swim tests were used to evaluate mice behavior on the 7th day after the last MPTP treatment.Tyrosine hydroxylase(TH)positive neurons and fibers of mice were determined by immunohistochemical staining.Results: The mice with MPTP treatment showed significant decrease total distances,rearing,cross numbers and swim scores while increase time to descend the floor in pole test compared with their vehicle counterparts.The mice with MPTP treatment showed significant decrease the number of TH positive neurons and fibers compared with their vehicle counterparts.Conclusion: The subacute MPTP mouse model can mimic some of behavior impairments of PD.Meanwhile,the dopaminergic neurons and fibers’ loss in subacute MPTP mouse model are in accordance with the typical pathological changes of PD.Section 2 The effect of melatonin on behavior and pathology in subacute MPTP mouse model of PDObjective:To investigate the effect of melatonin on behavioral and pathological indexes in subacute MPTP mouse model of PD,further identify the neuroprotective effect of melatonin on dopaminergic neurons.Methods:C57BL/6 mice were randomly divided into five groups:NS+ETH group,M30+NS group,MPTP+ETH group,M10+MPTP group and M30+MPTP group.MPTP mouse model of PD was established.The subacute MPTP-lesioned mice were treated with two doses of melatonin(10-30 mg/kg).Melatonin was freshly prepared in5% ethanol and administered at 30 min prior to MPTP,then three doses of melatonin,at every 1 hour of interval.Open-field,pole and swim tests were used to evaluate mice behavior on the 7th day after the last MPTP treatment.Then,mice were decapitated under anesthesia and their substantia nigra and striata tissues were rapidly removed.Western blotting analysis was used to examine the protein level of TH.Immunohistochemical staining was used to measure number of TH positive neurons and fibers.Results: Melatonin significantly improved MPTP-induced behavioral deficits in openfield test,pole test and swim test.Melatonin suppressed the reductions of nigral dopaminergic neurons and striatal fibers in mice with MPTP treatment.The mice with melatonin and MPTP co-treatment showed significant increase TH expression in the substantia nigra and striatum compared with MPTP+ETH group.Conclusion: Melatonin can improve the behavioral indexes and rescue the loss of dopaminergic neurons and fibers in subacute MPTP-lesioned mice.Section 3 The potential mechanism of melatonin in MPTP mouse model of PDObjective:To investigate the neuroprotective effect of melatonin in MPTP-induced mice model of PD and the possible mechanisms involved.Methods:C57BL/6 mice were randomly divided into five groups:NS+ETH group,M30+NS group,MPTP+ETH group,M10+MPTP group and M30+MPTP group.MPTP mouse model of PD was established.The subacute MPTP-1esioned mice were treated with two doses of melatonin(10-30 mg/kg).Melatonin was freshly prepared in 5% ethanol and administered at 30 min prior to MPTP,then three doses of melatonin,at every 1 hour of interval.On the 7th day after the last MPTP treatment,mice were decapitated under anesthesia and their substantia nigra tissue were rapidly removed.RT-PCR was used to examine the expression levels of i NOS,CD86,CD206,Arg1,IL-1β and IL-6 m RNA in the substantia nigra of mice.Western blotting analysis was used to examine the protein levels of i NOS,IL-6,p-P38,P38,p-ERK,ERK,p-JNK and JNK in the substantia nigra of mice.Iba-1 immunohistochemical staining was used to measure microglial activation in the substantia nigra of mice.Commercially available kit was used to measure the level of MDA in the substantia nigra of mice.Results: The mice with melatonin and MPTP co-treatment showed significant decrease the number of iba-1 positive cells in the substantia nigra compared with MPTP+ETH group.Compared with MPTP+ETH group,melatonin and MPTP co-treatment significantly decreased the expression levels of i NOS,CD86,IL-1β and IL-6 m RNA in the substantia nigra of mice,while increased the expression levels of CD206 and Arg1 m RNA.The mice with melatonin and MPTP co-treatment showed significant decrease the protein levels of i NOS,IL-6,p-P38/P38,p-ERK/ERK and p-JNK/JNK in the substantia nigra compared with MPTP+ETH group.Compared with MPTP+ETH group,melatonin and MPTP co-treatment significantly decreased MDA content in the substantia nigra of mice.Conclusion: Our results indicate that melatonin may exerts potent anti-inflammatory and antioxidative effects through selective modulation of microglia activation,thus exerting dopaminergic neuroprotection.