Study Of The Effect Of Exogenous Melatonin On MPTP Mice Of Parkinson' Disease

ObjectiveParkinson'disease (PD) is a central neurodegenerative disorder in the elderly affecting primarily the dopaminergic neurons in the substantia nigra (SN) and the nigrostriatal pathway, characterized by the clinical symptoms such as resting tremor, muscle rigidity, movements decrease, bradykinesia and posture imbalance.Now,the main clinical therapy is levodopa replacement to compensate for dopamine (DA).Even if it can improve the symptoms of PD,but the effect of these drugs on most patients is reduced after a period of administration,presenting some side effects such as dyskinesia, mental deterioration and psychiatric symptoms.Recent researches show that the pathogenesis of PD is involved in the oxidative stress induced by the increase of free radicals generation and the decrease of free radicals scavenging.Melatonin is a indole hormone secreted by the pineal gland and is a natural strong antioxidant.Considerable domestic and abroad experimental studies implicated that melatonin had a protective effect on the dopaminergic neurons, so it could ease the symptoms of PD.But it did not consider whether the circadian endogenous melatonin could influence its antioxidant effect.So, in this study, we gived mice exogenous melatonin at the maxmum peak(from 23 hous to 2 hous) and minimum peak(12 hours) of melatonin secretion respectively,then compared the effecs on the dopaminergic neurons in these two points to explore whether the endogenous melatonin could affect the protective effect on the dopaminergic neurons of exogenous melatonin.Methods1-Methy4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a fat-soluble neurotoxin, it can pass the blood-brain barrier easily and damage the nigral dopaminergic neurons selectively to induce the similar PD symptoms.Thus, this study used MPTP to establish the PD mouse model.60 healthy adult C57BL/6 mice were selected,male,10-12 weeks, ranged in weight from 250 to 350 g and supplied with food and water ad libitum with a 12-h light/dark cycle at room temperature.After 3 days,these mice were randomly divided into four groups:normal control group (5 mice), PD group (15 mice), MPTP+MEL night experimental group (abbreviated night group) (20 mice) and MPTP+MEL day experimental group (abbreviated day group)(20 mice).All the C57BL/6 mice in the PD group, night group and day group were intraperitoneally injected with MPTP(0.2ml/only,30mg-kg-) once a day, for 7 days.At the same time,the mice in the normal control group were intraperitoneally injected with normal saline 0.2ml/only once a day, for 7 days.On the eighth day,the mice in the day group and night group were intraperitoneally injected with melatonin 0.1 ml/only once a day, for 7 days. At the same time, the mice in the normal control group and PD group were intraperitoneally injected with normal saline 0.1 ml/only, once a day, for 7 days.Then,the mice were anesthetized conventionally,perfused and fixed through heart.Brain were removed and sectioned into 7μm thick paraffin slices.Tyrosine hydroxylase(TH) is the rate-limiting enzyme of the synthesis of DA in the brain.So,TH immunohistochemical staining,Western blot,immunofluorescence analysis and immune electron microscopy were used to compare the dopaminergic neurons in the SN and striatum.ResultsCompared with the normal control group,the amount of TH immunoreactive neurons,the expression of TH protein, the intensity of immunofluorescence, the immune deposits on the membrane of endoplasmic reticulum and electron density were significantly decreased in the PD group, but the day group had no apparent change.Compared with the PD group, the amount of TH immunoreactive neurons,the expression of TH protein, the intensity of immunofluorescence, the immune deposits on the membrane of endoplasmic reticulum and electron density were significantly increased in the day group, but the night group had no apparent change.There was no significant difference between the day group and night group. Conclusions1,Compared with the normal control group, the amount of TH immunoreactive neurons,the expression of TH protein, the intensity of immunofluorescence, the immune deposits on the membrane of endoplasmic reticulum and electron density were significantly decreased in the PD group, which implies MPTP is toxic to C57BL/6 mice and the model is established successfully.2,Compared with the PD group, the amount of TH immunoreactive neurons,the expression of TH protein, the intensity of immunofluorescence, the immune deposits on the membrane of endoplasmic reticulum and electron density were significantly increased in the day group, which indicates exogenous melatonin is neuroprotective to the dopaminergic neurons.3,The night group had no significant difference compared with the PD group, but were significantly increased compared with the night group, which indicates the antagonistic effect of endogenous melatonin on DA may affect the the dopaminergic neuroprotection of exogenous melatonin.