Investigation Involving The Production Of Rotenone Dopaminergic Destruction Model With Chronic Subcutaneous Injection And Its Mechanisms

Background and objective:Parkinson disease(PD) is a progressive neurological disorder with a prevalence of 1-2%in people over the age of 50.It has a world-wide distribution and has no gender preference.PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of intracytoplasmatic inclusions known as Lewy bodies.The selective degeneration of the nigrostriatal dopaminergic pathway is a fundamental characteristic in Parkinson disease(PD). Although the etiology of PD remains unclear,several lines of evidence indicate that living in a rural environment,farming,drinking well water and occupational exposure to agricultural chemicals are risk factors for developing PD.Considerable evidence suggests a multifactorial etiology involving genetic and environmental factors.In recent years,an increasing number of experimental evidence shows that the ubiquitin-proteasome pathway(ubiquitin proteasome pathway, UPP) dysfunction and the occurrence of Parkinson's disease are closely related.Ubiquitin-proteasome pathway includes ubiquitin,ubiquitin-conjugating enzyme(E2),ubiquitin-activating enzyme(E1),ubiquitin ligase (E3) and the 26S proteasome.Ubiquitin-proteasome pathway is very complex.A ubiquitin protein comprise76 amino acids which contas seven internal lysine residues(K6,K11,K27,K29,K33,K48 and K63).These lysine acid residues are the connection site of polyubiquitin chain extension. Ubiquitin's molecular weight is 40～50kD.Before the degradation of the protein,they must be marked by polyubiquitin chain.A problem of proteasome degradation may lead to the accumulation of abnormal protein and would therefore lead to production of Lewy bodies in dopaminergic neurons.Immunohistochemistry results showed thatα-synuclein is the main ingredient contained in this inclusion.α-synuclein is widely distributed in healthy brain tissue.The soluble protein includes 140 amino acids,is mainly located in the presynaptic neuron terminal areas.Under physiological conditions,α-synuclein may be to maintain the normal function of synapses,and involve in the regulation of dopamine biosynthesis.When injury occurs,α-synuclein aggregation may be adopted to resist injury.With theα-synuclein aggregation,abnormal proteins can not be cleared in time.Instead,they have a toxic effect on cells and accelerate cell death.At present,PD is divided into drugs treatment,neuroprotective treatment,as well as gene therapy treatment and neurological surgery.To date symptomatic treatment of Parkinson's disease is still the main means of treatment.It is difficult to prevent or slow disease progression.In addition, long-term symptomatic treatment will bring a lot of complications. Therefore,to further explore the pathogenesis and prevention and treatment of Parkinson's disease remains serious problems.Neurological disorders in humans can be modeled in animals using standardized procedures that recreate specific pathogenic events and their behavioral outcomes.The development of animal models of PD is important to test new neuroprotective agents and strategies.Such animal models of PD have to mimic,at least partially,a Parkinson-like pathology and should reproduce specific features of the human disease.Over the years,a broad variety of experimental models of PD were developed and applied in diverse species.Most PD(approximately 90%) is sporadic(sPD).Familial Parkinson's disease accounts for only 5%～10%of the total number. Moreover,recent research suggests that pathogenesis of sporadic PD may be related to mitochondrial complexⅠ.Therefore,Parkinson's disease animal model with inhibition of mitochondrial complexⅠshould be the best model.Recently,numerous in vivo and in vitro studies demonstrated that the herbicide rotenone produces specific dopaminergic cell degeneration accompanied by the formation of alpha-synuclein fibrils,cytoplasmic inclusions similar to those found in PD patients.Alpha-synuclein is a constituant of Lewy bodies,the hallmark of idiopathic Parkinson's disease. The destruction of dopaminergic neurons by rotenone is attributed to an inhibition of the neuronal mitochondrial complexⅠ.Rotenone is a natural pesticide in vegetables,fruit trees and medicinal crops.It is in a wide range of applications.Chronic exposure of rotenone can cause environmental pollution.Because rotenone is high lipophilic,it can go easily through the blood-brain barrier.Then it will act on brain mitochondrial respiratory chain complex(ComplexⅠ).The delivery of respiratory chain in cells will be blocked.Rotenone is different from other's complexⅠinhibitor,it can play a role in its toxicity directly without the need of the dopamine transporter,administration of rotenone in rats also occur Parkinson's disease-like catalepsy clinical Performance. Therefore,rotenone may be one of the potential pathogenesis of Parkinson's disease.Well,as soon as possible to establish a stable Parkinson model of animal studies will be of great significance to understand the role of the environmental factors in pathogenesis of Parkinson's disease In recent years, many researchers established rotenone rat models of Parkinson's disease.Due to complex experimental conditions and high mortality,it is difficult to promote large-scale application,so the purpose of our experiment is to improve the rotenone rat model.Methods:Sprague-Dawley male rats(180-220 g) were provided.The animals were divided in the follow groups:(1)Gradient dose model group(n=40), animals were first infused with 2mg/kg rotenone for 2days,followed by rotenone 1mg/kg dosing four days,the last dose of 0.5mg/kg rotenone for 22 days.(2)Constant dose rotene group(n=20),animals were infused with 0.5mg/kg dose rotenone for 28 days.(3)Vehicle group(n=14),animals were infused with dimethyl sulfoxide(DMSO) for 28 days.(4)Saline control group(n=6):animals were infused with saline for 28 days.To observe the general state and to get body weight Once a week.Before killing the rats,behavioral experiments were enforced.Contents of dopa- mine and its metabolites in striatum were detected by HPLC method Tyrosine hydroxylase(TH) immunohistochemistry in substantia nigra was carried out.heart,liver,spleen,kidney were observed by HE staining.The morphological changes of hippocampus and parietal cortex were observed by.Nissl staining.Protein contents of ubiquitin andα-synuclein in the substantia nigra and striatum were detected by Western blot method.At the same time,TH/ubiquitin and TH/α-synuclein immunofluorescence double staining were exerted.Results:1.The rats in gradient dose rotenone group had poor general state. Body weight was reduced daily.Behavioral obstacles appeared.Results of HPLC test showed that the dopamine in striatum was decreased more than 80%.Dopamine metabolites also decline.Results of tyrosine hydroxylase immunostaining in substantia nigra also showed that the number of positive neurons and nerve fibers decreased significantly.Rats in constant dose model group had no behavioral changes.Contents of dopamine and its metabolites were in normal level,but injury appeared in the substantia nigra dopaminergic neurons.2.HE staining results showed that no difference could be seen in heart, liver,spleen,kidney.No changes happened in parietal cortex and hippocampi.The comman staining including tyrosine hydroxylase immunostaining and nissle staining in the substantia nigra results showed that nissl bodies were no difference in brain areas outside the substantia nigra part in different groups.3.Results ofα-synuclein/ubiquitin western-blot detection in substantia nigra and striatum showedα-synuclein/ubiquitin expression were increased in substantia nigra from two model groups.no difference in striatum could be appeared between groups.The results ofα-synuclein/ ubiquitinand tyrosine hydroxylase immunofluorescence double staining were same to western-blot detection.Conclusion:1.The subcutaneous injection method with low-dose rotenone successfully established Parkinson's disease rat model. 2.To reduce the rotenone dose could significantly improve dopaminergic neurons in substantia nigra and animal behavior.3.Subcutaneous injection with low-dose rotenone could selectively induce dopamine neurons damages in substantia nigra.4.The total contents ofα-synuclein were increased in substantia nigra from two model groups.It showedα-synuclein aggregation could help to damage dopamine neurons.5.Expressions of ubiquitin and ubiquitin protein were increased in substantia nigra from two model groups.It showed ubiquitin-proteasome system dysfunction was related to dopaminergic neuron damage.