| Parkinson’s disease (PD) is a common progressive neurodegenerative disease of the central nervous system, characterized by bradykinesia, resting tremor, muscle rigidity and gait abnormalities. The incidence and prevalence increase year by year in PD along with the process of the aging population continues to accelerate. PD is the second prevalent neurodegenerative disorder after Alzheimer’s disease(AD) of the central nervous system. It not only impacts seriously on daily living and work ability of the elder, causes a heavy economic burden to the family and society, but also affects seriously the quality of life of older persons.Etiology and pathogenesis of PD are still not very clear until now. Studies suggest that PD may be associated with aging, environmental factors and genetic factors, Its pathogenesis are also involved in a variety of free radicals, oxidative stress, excitatory glutamate toxicity, neurotrophic factor deficiency, chronic neuroinflam mation, mitochondrial complex I deletion and apoptosis, the interactions of these mechanisms ultimately lead to the neuronal death in complex cascade of biochemical reactions. But the exact pathogenesis is unclear. At present, the purpose for effective therapy is as long as possible to improve function of patient. But we can not prevent the development of neurodegenerative processes in PD, and there may be some serious treatment-related complications in the late, it is still an incurable disease. Therefore, more effective prevention and treatment are needed.We observed nonsteroidal anti-inflammatory drugs could reduce long-term risk of Parkinson’s disease in epidemiological data. Currently, accumulation of activated microglia could be observed around the degenerative neurons in many neuroinflam mation and neurodegenerative diseases. It is important in the pathogenesis of PD due to excessive activation of microglia lead to the occurrence of inflammation and oxidative stress and damage dopaminergic neurons. Neuroinflammation is considered a key pathogenesis in the progression of many neurodegenerative diseases including AD and PD. Several studies have found that activated microglia can release neurotoxicity or proinflammatory cytokines, including NO, TNFa, PGE2, IL-1β, and IL-6. These pro-inflammatory cytokines can deteriorate neuronal injury via receptor-dependent apoptotic pathway in nerve inflammation process. Therefore, an effective therapeutic strategy may has the neuroprotective effect by means of inhibition inflammatory reaction including microglial. However, microglia activation pathway and the role of release of inflammatory cytokines in the pathogenesis of Parkinson’s disease are still in the debate. Toll-like receptors (TLRs) are a class of cell surface signaling transmembrane receptors, belong to class I transmembrane receptors. They not only can identify the pathogenic microorganisms in the natural immune system, activate the innate immune response, are the main receptors of natural immune system to recognize pathogenic microorganisms, but also can identify the pathogen by antigen presenting cells, start the adaptive immune response by generating costimulatory signal, they are the bridge between innate immunity and acquired immunity in our body. Existing research suggested that TLRs associated with neurodegenerative diseases. Miguel etc also suggested that TLR4 induced microglial activation. Therefore, we believe that:a neuroprotective effect for inhibiting the release of microglia activation-induced inflammatory mediators plays a far more important role than protecting the neurons themselves.More sufficient evidence proved neuroinflammation as an important pathogenesis of Parkinson’s disease, microglia activation and release of inflammatory mediators played a key role. According to the latest research theories we supposed that TLR4 may be the key triggered receptor in neuroinflammation pathways. So we assumed inhibit TLR4 expression, may be could inhibit the activation of microglia and the subsequent release of inflammatory mediators, thereby block the occurrence or development of PD partially? Based on this theory, we designed to detect the content of TLR4,dopaminergic neurons and microglia in the classical mouse models of PD and detect the expression of inflammatory mediators such as TNF-a and IL1β, etc, then inhibit the expression of TLR4 by TLR4-specific antibodies and analyze the expression of inflammatory mediators such as TNF-a and IL1β,and contrast the behavior, numbers of dopaminergic neurons and microglia activation state in nigrostriatal of mice between anti-TLR4+MPTP group and MPTP group at the same time. Eventually reveal that whether TLR4 is a key receptor of neuroinflammation in neurodegenerative diseases, and delay the progression or relieving symptoms in mice with Parkinson’s disease, and whether can predict the onset and progression of PD by early detection of activated microglia or TLR4 receptor.Chapter 1 Establishment of the MPTP-induced Parkinson’s disease mouse model and its evaluation methodIt is essential for the study of Parkinson’s disease that a stable Parkinson animal model and a efficient evaluation method. Animal models of Parkinson’s disease can be divided into three categories:A:agent-induced model, B:gene model, C: dopamine neuron-specific gene knockout model. Although these models have advantages and disadvantages, but no single model can fully replicate all the features of Parkinson’s disease due to its complex clinical symptoms. The most common model in PD clinical research is the model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but the evaluation methods are different. To further establish the stable PD animal model and efficient evaluation method, forty-eight C57BL/6J mice were divided into three groups randomly, normal control group (group 1),MPTP group (group 2), TLR4 antibody+ MPTP group (group 3), respectively,and sixteen mice in each group. The mice were injected intraperitoneal once a day with either 30 mg/kg MPTP(group2 and group3) or a corresponding volume of saline(groupl) seven times. Every time after injection, mice were observed in all groups whether appeared involuntary tremors, salivation, slow, postural instability, stiff limbs, erect hair or tail and other short-term symptoms, and whether declined in overall initiative, including the ability of eating and activities, etc., and bradykinesia, unsteady gait, clumsiness, bending arched, uncoordinated movement in paws and other persistent symptoms. To allow mice to adapt to laboratory conditions, try to avoid interference the experimental results by other factors, the mice were moved to behavioral testing room from breeding room in two hours before the experiment. In order to minimize experimental error due to the speed of learning, mice were trained on mice drum instrument in 2 days before making Parkinson’s disease models. Mice drum instrument was used to evaluate motor coordination of mice. Training test were operated twice a day, speed was 12r.min, training time was 120s, the time of each mouse were recorded from the drum starts rotating to leave the drum as the mouse movement falls incubation period. Formal experiments of mice drum instrument test were operated in one day after the administration. The speed was 24r.min, training time was 360s in formal experiments. Each mouse was measured three times and averaged in falls incubation period. Eight mice were randomly selected to cardiac perfusion fixation and taken all the brain in each group for detecting pathological morphology and the number of TH-positive cells in midbrain after all the tests had been completed. Data were expressed as mean±standard deviation. The Statistical analysis software was SPSS16.0.Statistical analysis was performed using one-way ANOVA with expression of TH-positive dopaminergic neurons in midbrain of mice and drop incubation period of mice among three groups if the error variance of the dependent variable was equal across groups, the dependent variable of each groups were compared using Bonferroni method. Welch robust test was performed if the error variance of the dependent variable was not equal,the dependent variable of each groups were compared using Tamhane’ T2 method..p<0.05 was considered to be statistically significant.In two minutes after the injection of MPTP, C57BL/6J mice began to appear restless, running back and forth in the cage or climb, chin resting tremor, nodding like tremors, piloerection reactions. About 10 minutes after the injection start to curl up into a group, less exercise, scatter fur, eyes dull, vertical hair, arch, vertical tail and reduce reaction to external stimuli or no response. All the acute toxic reactions began to weaken after about four hours, disappeared after 24 hours, but the movement was still reduced.In five days after the beginning of the injection of MPTP, the mice began to decline in the overall initiative, including the ability to eat and activities, and bradykinesia, unsteady gait, clumsiness, bending arched, uncoordinated movement in paws and other persistent symptoms. Objective and quantitative detection of motor coordination showed that the drop incubation period in mice was shorter in MPTP group than TLR4 antibody +MPTP group, and longest in control group,the drop incubation period of mice among three groups were significant (F=970.707, P <0.001). It suggest that the movement coordination capacity was significantly impaired in MPTP-induced Parkinson’s disease mice models. TH-positive dopaminergic neurons in Nigra of mice among three groups were different significantly (F=97.406, P<0.001), and a significant difference between each group by comparison with each other (P<=0.001).TH positive dopamine neurons increased significantly in control group, followed by TLR4 antibody + MPTP group, and a minimum in MPTP group. Smaller dopaminergic neurons, lighter cytoplasmic staining, decreased number of cells and shorter projections were observed at high magnification in MPTP group. In summary, our data demonstrate that clinical manifestations of MPTP-induced subacute PD models in mice were similar to that of PD patients, the dopaminergic neuron loss in midbrain significantly were showed by immunohistochemistry, and all these features were comply with the requirements of the PD models in animals. The motor symptoms of PD animal models can be evaluated more objectively by mice drum instrument test.Chapter 2 Expression of TH, TLR4, CD11 b and inflammatory mediators in the midbrain of PD mice models and impact on their expression by TLR4 antibodiesTo further verify the role of TLR4 receptors in the pathogenesis of Parkinson’s disease in mice and impact on the expression of TH, TLR4, CD11b and inflammatory mediators by TLR4 antibodies, we were still in accordance with the experimental program of the part 1. Forty-eight C57BL/6J mice were divided into three groups randomly, normal control group (group 1),MPTP group (group 2), TLR4 antibody+ MPTP group (group 3) respectively,and sixteen mice in each group. The mice were injected intraperitoneal once a day with either 30 mg/kg MPTP(group2 and group3) or a corresponding volume of saline(groupl) seven times. Since the objective existence of the blood brain barrier(BBB), so we should consider the BBB when administered TLR4 antibody to mice. The current view is that administer by nasogastric or intracerebroventricular injection are two simple way easily through the blood-brain barrier. Despite the extensive literature showed that it did not guarantee the drug concentration in the brain by nasal administration,we still conducted preliminary experiment in accordance with the above two methods reported in the literature, and tested mice by the In-Vivo FX PRO automatic preclinical small animal imaging system to confirm the stability and effectiveness of the two routes of administration. The results from the vivo imaging of mice showed that there almost no red fluorescence in lateral ventricles of C57BL/6J mice after nasal administration Anti-Mouse CD284 (TLR4) PE nasal, suggest nasal administration is poor efficacy, does not guarantee effective drug concentration.There were red fluorescence in bilateral ventricle by injection of Anti-Mouse CD284 (TLR4) PE in the right lateral ventricle, Confirm that the lateral ventricle administration can ensure effective drug concentration in the central nervous system. We chose intracerebroventricular injection TLR4 antibody owing to the mice recovery sooner after the injection, had no significant acute injury response,easy to operation and practical. Fluorescently labeled monoclonal antibodies of TLR4 were injected intraventricular once a day with 20ug in two days before the injection of MPTP in group 3 or a corresponding volume of saline (group1and group2) two times. In order to minimize experimental error due to the speed of learning, mice were trained on mice drum instrument in 2 days before making Parkinson’s disease models.Formal experiments in mice drum instrument test were operated in one day after the administration.Eight mice were randomly selected to been decapitated quickly on the ice to take a fresh brain tissue to detect the mRNA expression levels of TLR4, TNFa and IL1β in the brain.Another mice were selected to cardiac perfusion fixation and take all the brain for detecting pathological morphology and the positive cells number of TLR4,TH and CD 11b in the midbrain in mice. Data were expressed as mean±tandard deviation. The Statistical analysis software was SPSS16.O.Statistical analysis was performed using one-way ANOVA with expression of TLR4mRNA, TNFamRNA, IL1βmRNA, TLR4-positive cells,CD11b-positive cells and TH-positive dopaminergic neurons in midbrain of mice and drop incubation period of mice among three groups if the error variance of the dependent variable was equal across groups, the dependent variable of each groups were compared using Bonferroni method. Welch robust test was performed if the error variance of the dependent variable was not equal,the dependent variable of each groups were compared using Tamhane’ T2 method.P<0.05 was considered to be statistically significant.TH-positive dopaminergic neurons in midbrain of mice among three groups were different significantly (F= 97.406, P 0.001), and a significant difference between each group by comparison with each other (P<=0.001).TH positive dopamine neurons increased significantly in control group, followed by TLR4 antibody + MPTP group, and a minimum in MPTP group. It confirmed that the PD mice models were modeled successfully and the intervention by TLR4 antibody were effectively. There were significant differences among three groups regarding the number of CD 11b-positive cells in mice in midbrain (F=131.856, P 0.001), and a significant difference between each group by comparison with each other (P<0.001). CD11b-positive cells increased significantly in MPTP group, followed by TLR4 antibody + MPTP group, and a minimum in control group.It suggested that MPTP induced microglial activation and TLR4 antibody was able to partially reduce this activation.There were significant differences among three groups regarding number of TLR4 positive cells in the brain (F=331.758, P<0.001), and a significant difference between each group by comparison with each other (p<0.001).TLR4 positive cells increased significantly in MPTP group, followed by TLR4 antibody+ MPTP group, and a minimum in control group.Expression of TLR4mRNA, TNFamRNA and IL1βmRNA in midbrain were different significantly in three groups (F=36.385, P<0.001; F=299.599, P<0.001; F=40.191, P<0.001 respectively), there were significant differences among three groups regarding expression levels of TLR4mRNA and IL1βmRNA by comparison with each other (P<0.01), and there were significant differences among three groups regarding expression levels of TNFα mRNA by comparison with each other (P<0.05), expression increased significantly in MPTP group, followed by TLR4 antibody+MPTP group, and a minimum in control group.It suggested that MPTP induced downstream massive release of pro-inflamma tory factors, and TLR4 involved in the pathological process, TLR4 antibody could reduce this pro-inflammatory cytokine release. Therefore, we conclude that TLR4 may be the key receptor which trigger neuroinflammation pathways in neurodegene rative disease and TLR4 receptor trigger the activation of microglia and induce the release of downstream pro-inflammatory cytokine, eventually lead to the death of dopaminergic neurons.Specific TLR4 antibody can partially block MPTP-induced neuroinflammatory process, mitigate the activation of microglia, reduce the expression of pro-inflammatory cytokines such as TNFα and IL-1β, the death of dopaminergic neurons,therefore, partially mitigate abnormal behavior of mice models of Parkinson’s desease. Because our sample size is smaller, further expand sample multi-center study are needed to reveal whether pharmacological modulations in the TLR4-pathway could represent a novel therapeutic approach in the treatment of PD.In summary, our work demonstrate that clinical manifestations of MPTP-induced subacute PD models in mouse are similar to that of PD patients, the dopaminergic neuron loss in midbrain significantly are showed by immunohisto chemistry, and all these features are comply with the requirements of the PD models in animals. The motor symptoms of PD animal models can be evaluated more objectively by mice drum instrument test.TLR4 may be the key receptor which trigger neuroinflammation pathways in neurodegenerative disease and TLR4 receptor trigger the activation of microglia and induce the release of downstream pro-inflam matory cytokine, eventually lead to the death of dopaminergic neurons.Specific TLR4 antibody can partially block MPTP-induced neuroinflam matory process, mitigate the activation of microglia, reduce the expression of pro-inflammatory cytokines such as TNFα and IL-1β, reduce the death of dopaminergic neurons,therefore, partially mitigate abnormal behavior of mice models of Parkinson’s desease.Further studies will reveal whether pharmacological modulations in the TLR4-pathway could represent a novel therapeutic approach in the treatment of PD. |
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