Echinacoside Rescues Behavior Impairments And Deficits Of Nigrostriatal Dopaminergic Pathway In Subacute MPTP Mouse Model Of Parkinson's Disease

Parkinson's disease (PD) is one of common neurodegenerative diseases, which is characterized by a progressive and selective loss of nigral dopaminergic neurons, resulting in a pronounced depletion of striatal dopamine. The loss of striatal dopamine and the consequent dysfunction of the nigrostriatal pathway lead to a range of motor symptoms, including resting tremor, rigidity, bradykinesia, gait disorder and loss of postural reflex. As the disease progresses, many patients also develop some cognitive dysfunction, including anxiety, depression and dementia. In contrast to other neurodegenerative disorders, there are relatively good symptomatic therapies available for the treatment of Parkinson's disease. These therapies consist predominantly of dopamine replacement and adjuvant surgical therapy to relieve most of the motor symptoms of the disease. However, these symptomatic therapies are not themselves without problem, for example, long-term treatment with the dopamine precursor 3,4-dihydroxyl-phenylalanine (L-DOPA) often leads to the development of debilitating dyskinesias and it can not stop the progression of PD. Furthermore, at the present time, there is no disease-modifying neuroprotective or neuroresue/neurorestorative therapy available.Recently to stimulate the endogenous expression of neurotrophic factors (NTFs), especially glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) shows a petency to prevent or halt the progress in PD. According to some results of our previous researches, we hypothesized that oral administration of ECH, a monomer extracted from herbs cistanchis, to subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, commencing after impairment of the nigrstriatal system, had neurorescue effects on both behavior,pathological and neurochemistrical deficits meanwhile attenuated the factors correlated with survival or death of dompaminergic neurons, such as GDNF and BDNF, Bcl-2 family members and endoplasmic reticulum stress mediator CHOP at both mRNA and protein levels. Objective To investigate the sensitive and stable behavior tests for the subacute MPTP mouse model of PD, find further verifications by methods of pathology and neurochemistry.Methods Observe behavior changes of C57BL/6J mice before MPTP intraperitoneal injection and 1,3,5,7,14 days after the last administration of MPTP (30mg/kg/day×5 days) including open field activity, rotarod test, pole test, gait analysis, grid test and adhesive removal test. After the last observation mice were sacrificed for TH immunohistochemistry detection and dopaminergic neurotransmitter detection by HPLC-EC. Independent samples T-Test was performed for statistical purposes.A value of P<0.05 was considered statistically significant.Results 1.The MPTP-lesioned mice demonstrated obvious tremor and rigid symptoms several to 30 minutes after every intraperitoneal injection and recovered in few hours. For MPTP administrated mice the distances of horizontal movement, rearing numbers, latency to fall in rotarod test, stride length of fore-and hindlimb decreased while the time to remove adhesive tape and time to turn in pole test were both prolonged compared with the saline control mice within 7 days after the last MPTP injection. However some of index recovered spontaneously later and only stride length, rotarod behavior dysfuctions and rearing numbers kept abnormal 14 days after the last MPTP administration.2.Comparing with the saline control animal, the number of TH-ir neurons and fibers in MPTP mice decreased and the contents of DA and DOPAC reduced significantly 14 days after the last MPTP injection (P＜0.001).Conclusion 1.The subacute MPTP mouse model can mimic most of behavior impairments of PD including tremor and muscular rigid, akinesia, bradykinesia, gait abnormalities with poor postural balance.The gait analysis is the most sensitive and stable index for research of neuroresue drugs and the next choice are rotarod test and rearing numbers.2.The dopaminergic neurons and fibers'loss in subacute MPTP mouse model are in accordance with the typical pathological changes of PD and can be retained to 14 days after the end of leision.3.The reductions of DA and its metabolisms verify the model from the view of neurochemistry further and can also last to 2 weeks after the last MPTP injection.Objective To investigate the neurorescue effects of ECH in subacute MPTP mouse model of PD and the possible mechanisms involved. Methods The subacute MPTP-lesioned mice were treated with three doses of ECH (10, 20,30mg/kg/day) and selegiline (1.0mg/kg/day)for 14 days started at 24 hours after the last MPTP injection. Behavior assessments were performed at the 7th and 14th day of treatments according to the methods of part I on gait, rotarod and rearing. After the last observation of behaviors mice were sacrificed for detections of TH immunohistochemistry, dopaminergic neurotransmitters detection by HPLC and TUNEL apoptotic cell counts. The mRNA and protein levels of GDNF, BDNF, Bax, Bcl-2 and CHOP in substantia nigra of mice were quantified by Real time PCR as well as Western blot techniques. One-way ANOVA followed by post-hoc analysis of Tukey's HSD and Student-Newman-Keuls multiple comparisons test was performed for statistical purposes. A value of P<0.05 was considered statistically significant.Results 1. At the 7th and 14th days of high-dose ECH treatment, the gait disorder displayed obvious improvement (P<0.05) meanwhile the rotarod and rearing behavior also had slight ameliorations compared with the saline treated MPTP mice, which is similar with selegiline treatment. The behaviors in other two doses of ECH treated mice were improved to some extent (P>0.05).2.The high doses of ECH suppressed the reductions of nigral dopaminergic neurons (P<0.001) and striatal fibers (P＜0.01), contents of DA (P<0.05) and its metablisms meanwhile decreased the number of apoptotic cells compared with saline treated MPTP-lesioned animals (P<0.001). There was a relative elevation in expression of GDNF and BDNF mRNA (2.94 and 3.75-fold) and proteins (184.34% and 185.93%) in high dose of ECH treated mice compared with vehicle treated MPTP-lesioned mice. In addition, the Bax/Bcl-2 ratio as well as gene and protein of CHOP in MPTP-lesioned mice significantly increased, and these effects could be prevented by ECH. The moderate dose of ECH and selegiline had similar but weaker effects.Conclusion 1.The high dose of ECH improves the gait abnormal with poor postural balance and akinesia in the subacute MPTP-lesioned mice.2.ECH can rescue the loss of dopaminergic neurons, fibers and DA after the MPTP lesions in certain dose-dependent manner then exhibits similar activites with selegiline. So ECH demonstrates a potency in the disease-modifying and symptom treatments of PD.3.ECH up-regulates pro-survival factors GDNF and BDNF, meanwhile has anti-apoptotic effects through attenuation to Bcl-2 family members and CHOP which indicates the mitochondria and endoplasmic reticulum pathway are probably involved.