| Parkinson's disease (PD) is a common neurodegenerative disorder in the population over age 50. It already becomes the one of diseases causing death and disability is increasingly common with advanced age. A major pathological hallmark of PD is a progressive loss of dopaminergic neurons in the substantia nigrapars compatcta(SNpc) and their axons, which project to the striatum. The loss of dopaminergic neurons results in decrease of dopamine in the striatum and imbalance in dopamine and acetylcholine. There is still no effective treatment because of the complexity of the etiopathogenisis and mechanisms of PD. In recent years, the neural stem cells research continued to deepen, and many scholars have tried to stimulate endogenous neural stem cells to differentiate into dopaminergic neurons, to supplement the lost cells and restore physiological functions. Chinese herbal medicine have efficacy at treating PD. From the perspective of stimulation of endogenous neural stem cells proliferation and differentiation, reveal interference effect and the mechanisms by using EGB in MPTP-treated PD model mouse. It provides new method to improve the ability of neural stem cells proliferation and differentiation of dopamine neurons, and lays foundation to cure PD by using neural stem cells, and supplies scientific and experimental basis in PD therapy applying Chinese medicine.This study is consisted of three parts: (1)To observe the ethological and histopathologic changes in MPTP-treated PD model mice, meanwhile observe the effect of EGB; (2) To observe the proliferation and differentiation of NSC in brain in Parkinsonian mice and the influences of EGB by immunohistochemistry and double immunohistochemistry techniques. (3)To observe the expression of the protein Nurr1 which is related to NSC developed into dopaminegic neurons, and effect of EGB.In experimental research, male C57BL/6 mice are injected intraperitoneally with a total cumulative dose of 150mg/kg MPTP ,30mg /kg daily to induce Parkinson's disease model. The modeled mice are called Parkinsonian mice. The experimental animals is divided randomly into three groups:(1)The normal control group( NC); (2) The model control group( MC);(3) Ginkgo Biloba Extract group( EGBG). The MC treated nothing after modeled. EGBG injected intraperitoneally with EGB 70mg/kg/d since induced with MPTP. The animals were killed respectively on the 7th and the 28th day after first injecting with MPTP.The main results are displayed as follow:(1)Effects of EGB on the ethological changes and the loss of dopaminegic neurons in MPTP-treated PD model mice.NC mice moved sensitively, acted cover a wide range, no tremor, in general climbed smoothly and no stay in a short time. All the mice showed behavioral disturbance after MPTP injecting, including acute and chronic changes. Especially in the first week, these changes displayed significantly. The time of pole-climbing is also prolonged in the first week. Compared with the NC, the difference is significantly. After that the symptom began to recover slowly, and the difference also not became obviously. We can see that neurons in the SNpc are eumorphism, volume is normal and structure is clearly in NC. On the 7 th day and 28 th day, the number of neurons in the SNpc is decreased obviously in MC. Compared with MC, the neurons increased a little at EGBG on 7th day, and there has no difference between MC and EGBG. But on 28 th day, the number of dopaminegic neurons increased significant.The immunohistochemistry of TH showed that, large number of TH positive neurons and fibers were concentrated in SNC and VTA in NC. The TH positive neurons and the value of IOD of MC and EGBG decreased obviously, survived TH+ neurons showed weakly positive on 7 th day. Compared with NC, there are significant changes in MC and EGBG. On 28 th day, the value of IOD of TH+ neurons in SNpc of EGBG is larger compared with MC.(2)Effects of EGB on proliferation of neuron stem cells in brain in PD mice.Normal HE staining was observed in the lateral angle and lateral ventricle bottom corner, there are some cells staining anachromasis in NC. These cells are ellipse or round and exist in group. On 7 th day, the number of cells of lateral angle, lateral ventricle bottom corner and lateral wall in lateral cerebral ventricle in MC were all increased. The cells increasing of these areas in EGBG were more than MC. On 28 th day, although cells of lateral angle, lateral ventricle bottom corner were increased obviously in MC and EGBG, there are no difference among MC and EGBG.The PCNA positive cells existed at the subependymal zone(SVZ) in lateral angle, lateral ventricle bottom corner and lateral wall in lateral cerebral ventricle in NC. On 7 th day, the number of PCNA positive neurons of EGBG was increased significant compared with MC. On 28 th day, in lateral cerebral ventricle, the PCNA positive neurons were varying increased in MC and EGBG, but the differences are not significant between these two groups. At hippocampal dentate gyrus, the PCNA positive neurons existed in the subgranule zone (SGZ). On 7 th day, there were no differences obviously in three groups. On 28 th day, compared with NC and MC on 7 th day, there were obviously increase in MC and EGBG. But EGBG increased not more than MC.The immunohistochemistry of Nestin showed us that little Nestin+ neurons exist at lateral angle, lateral ventricle bottom corner in lateral cerebral ventricle. On 7 th day, the Nestin+ neurons increased obviously in MC and EGBG, and EGBG more than MC. On 28 th day, the Nestin positive neurons varying decreased in lateral cerebral ventricle in MC and EGBG, but increased compared with NC. At hippocampal dentate gyrus, the Nestin+ neurons always exist at polymorphic layer. On 7 th day, the Nestin+ neurons increased obviously in MC and EGBG. There are significant difference among EGBG and MC, EGBG and NC. On 28 th day, the change of the Nestin+ neurons in MC is not obvious. The positive cells is decreased in EGBG on the contrary.The double immunohistochemistry of PCNA and Nestin showed that double positive neurons can be found in lateral angle, lateral ventricle bottom corner in lateral cerebral ventricle, and little cells exist at SGZ. Compared with NC, the cells has increased in MC and EGBG.(3) Effects of EGB on expression of Nurr1 in brain in PD mice.The immunohistochemistry of Nurr1 showed us that the positive cells can be seen at SNpc and hippocampal dentate gyrus in NC. On 7 th day, the value of optic density of Nurr1+ neurons increased obviously in MC and EGBG in these two areas, and EGBG more than MC. On 28 th day, the optic density of Nurr1 in EGBG was increased obviously, and has significant difference with NC and MC. MC has not has this change.Conclusions: (1) MPTP can induced the loss of dopaminegic neurons in SNpc. The ethological changes are obvious at prophase. EGB can defend the damage and death of neurons in SNpc in MPTP-treated PD mice. (2) The damage can stimulate the proliferation of neuronal stem cells in hippocampal dentate gyrus and lateral cerebral ventricle, and EGB can farther stimulate the proliferation. These cells probably can migrate to other areas. The Nurr1 protein is very concerned with the differentiation of dopaminegic neurons. EGB can increase the expression of Nurr1. This indicated that these increasing neuronal stem cells probably can different to dopaminegic neurons. |
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