| In the present, cerebrovascular disease has become one of the human primary death diseases, which are high prevalence, high morbidity, high mortality rate, and high recurrence rate. Since cerebral infarction accounts for about 85% of the cerebrovascular disease, research on prevention and treatment of cerebral infarction is of great significance. It was reported that the treatment of cerebral infarction is not effective. Neural dysfunction is a major cause of disability in patients with after cerebral infarction. The injury to neurite outgrowth of neurons is one of the important factors leading to nerve dysfunction. So, how to promote the neurite growth of neurons is the key to the treatment of cerebral infarction.After cerebral infarction, brain tissues suffer from different degree of damage, andthe structure of neural network is destroyed, which resulting in impaired nerve. Neural plasticity could prompt to form a new neural network, which can activate and repair the lesions of brain, and improve neural function of ischemic brain. So it is particularly important to study the drugs strengthening the function of nerve repair. Because the neurite outgrowth of neurons is of great significance to repair of neural network, looking for drugs effectively promoting the neurite outgrowth of the neurons is a hot topic and important.Sonic hedgehog(Shh), a secretory glycoprotein factor, can regulate the proliferation and differentiation of nerve cells and the formation of axons in the occurrence, development and differentiation of the embryo and nervous system. It has been reported that in ischemia rats, Shh can promote neuronal plasticity by adjusting the proliferation and differentiation of nerve cells and the formation of neurite outgrowth, and participate in the regulation of nerve regeneration of brain SVZ area. So far, the effect and its potent mechanism of Shh proteins on neurite outgrowth of neurons is still not clear, which still needs further study, and is of great significance to strengthen the study of the neural circuits.Oxidative stress is one of the important pathophysiological mechanisms of cerebral infarction. And it is helpful to understand the pathogenesis of cerebral infarction and to research the relevant treatment drugs using oxidative stress model. So this study used oxidative stress model to simulate the cortex neuron injury after cerebral infarction to study the protection of Shh proteins on the cortex neurons under oxidative stress.Mitochondrion is the main place of energy production, which may play an important role in control of neuroplasticity, neurite growth, neurotransmitter release, and dendritic remodeling. Mitochondrial dysfunction may is crucial to neural plasticity after cerebral infarction. Adjusting the mitochondrial morphology and function after cerebral infarction can induce the neurite outgrowth, which needs enough energy. Therefore, energy metabolism and mitochondria are highly relevant to neurite outgrowth. It is showed that activating classical Shh signaling pathway can increase the activity of the mitochondria. But about for, the influence of Shh on the cortex neuron mitochondria under oxidative stress was less reported.In conclusion, primary cortical neurons were obtained from the embryonic brains at E15-18 of C57BL/6 pregnant mice. Using primary cortical neurons model, the effect and the mechanism of Shh on neurite outgrowth was analysed. Lastly, we use cortex neurons under oxidative stress model to simulate the cerebral infarction. The effect of Shh onneurite outgrowth of cortical neurons under oxidative stress was analysised, the underlying mechanism on mitochondria and energy metabolism were evaluted. Part I The cultivation and identification of primary mousecortical neuronsObjective:To master the primary culture method of cortical neurons in mice using the embryonic day 15-18 of C57BL/6 mice, and observe the cell morphology; Using immunocytochemistry method to identify whether the cultured cells were neurons.Methods:The embryonic brains was obtained at embryonic day 15-18(E15-18) of C57BL/6 mice and placed into the HBSS in the ice. The cerebral cortex of the embryonic brains was isolated and the meanings of the cerebral cortex were removed under an optical microscope in HBSS buffer in the ice. The cerebral cortex were dissected in the ice and incubated in Hibernate-E containing 2.0mg/ml papain for 15 min at 37°C. The dispersed cerebral cortical tissues were then neutralized with Hibernate-E and dissociated into single cell in neurobasal medium containing 2% B27 supplement and 0.5mM glutamine with a 1 ml pipette. The dissociated neurons were plated onto culture dishes coated with poly-L-lysine and grown in neuronal culture medium containing 2% B27 supplement and 0.5mM glutamine and incubated in a humidi?ed 5% CO2 incubator at 37 °C. After 24 h cultures, primary cortical neurons were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization using 0.3% Triton-X-PBS for 15 min at room temperature. Nonspecific binding was blocked with 10% normal donkey serum diluted by 1% bovine serum albumin(BSA)(IgG free)(Sigma, USA) and 0.3% Triton- X-PBS for 45 min at room temperature. Primary cortical neurons were then incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 h at room temperature. The images were captured using an upright fluorescence microscope. The primary antibodies used in this study included: mouse monoclonal anti-NSE antibody, rabbit anti mouse GFAP. The secondary antibodies used in this study were donkey anti-mouse IgG, FITC, Conjugated(1:200) and donkey anti-rabbit IgG, TRITC, Conjugated(1:200, CWBIO, Beijing China).Results:1 The cortex neuron cells were round when just cultivating. After 2 hours, the cells began to stick and 6~8 hours later, neurites obviously generated in a small number of neurons. When cultrued 24 hours, the body of neurons were clear and the neurite outgowth wae increased. After 2 days, the body of cell growth grew more and the neurite outgrowth was longer. Some neurons formed a network structure after culturing 3 days.2 This experiment used immunofluorescence method to detect NSE-labeled neurons which mainly appear in the vision field. However, GFAP-labeled glial cells are not found in field of vision.Conclusion:1 This experiment using serum-free culture method successfully established the primary mouse cortical neurons culture model,, which is a kind of ideal model with high stability, high purity, and survival of cortical neurons for research of neuroscience.2 To cultivate of fetal mouse cortical neurons of The identification results by immunofluorescence method indicates this method for primary culture of cortical neurons is high survival rate and purity, laid a good foundation for subsequent experiments. Part II The effect of Shh on neurite outgrowth of cortical neuron and its mechanismObjective: The aim is to observe the effect and the possible mechanism of Shh protein on the neurite outgrowth of cortical neurons in mice.Methods: Primary cultured cortical neurons were obtained from E15-18 of C57BL/6 mice. The expriment was divided into control group, different concentrations of Shh group(5,50,500ng/ml), Shh signaling pathway inhibitor group(cyclopamine, CPM), the CPM+ Shh group and K252a+Shh group. Neurons were treated with different drugs for 24 h. To block the Shh pathway, CPM or K252 a was added 30 min before Shh treatment.The expression of Shh receptor(Ptch)was deteched by the immunofluorescence; The effect of Shh on neurite outgrowth of cortical neurons was determinated by β-Tubulin immunofluorescence staining; Using RT-qPCR and Western-bloting method to detect the expression levels Shh, Ptch and BDNF gene and protein in Shh-indeuced neurons; foring the content of BDNF on neuronal neurite was determined by using immunofluorescence cytochemistry staining method. The content of BDNF in the culture medium was determined by ELISA method according to the BDNF kit instructions.Results:1The receptor for Shh(Ptch)is expressed in primary cortical neurons, suggesting that Shh could have biological effects on primary cortical neurons to regulate neurite outgrowth.2 50 ng/mL and 500 ng/mL Shh significantly increased neurite outgrowth in primary cortical neurons compared to untreated control(P<0.05). In addition, Shh 50 ng/mL had a remarkable promoting effect.3 After co-administration of Shh and CPM, the neurite length was significantly reduced compared with Shh-treated group(P<0.05). However, there was no significant difference between the control group and CPM alone group. Moreover, significant difference existed between the Shh + CPM group and the control group(P<0.05). These data indicate that Shh-induced neurite outgrowth in primary cortical neurons is at least partially blocked by CPM.4 The expressions of Shh and Ptch genes were significantly increased in Shh-treated group, and decreased after treatment with Shh + CPM(P<0.05). However, the expressions of Shh and Ptch genes were not significantly different between the control group and CPM alone group. These results suggest that the inhibition of the Shh pathway partially blocks Shh-induced neurite outgrowth.5 Shh significantly increased BDNF gene expression and protein levels in primary cortical neurons, while blocking the Shh pathway decreased the BDNF gene and protein levels in primary cortical neurons(P<0.05).6 Quantification of BDNF released in the culture medium showed no significant difference between the control group and CPM alone group. However, the levels of BDNF in the culture medium were increased in the Shh-treated group and partially inhibited in the Shh + CPM group(P<0.05).7The levels of BDNF in the neurite outgrowth were increased in the Shh-treated group and partially inhibited in the Shh + CPM group(P<0.05).8 The effect of Shh on neurite outgrowth in primary cortical neurons was inhibited by K252a(P< 0.05).Conclusions:In summary, the results show that Shh protein could increase the neurite outgrowth of cortical neurons, partly by activating Shh signaling pathway, and partly by promoting the expression of BDNF. Part III The effect and the potent mechanism of Shh onneuroprotective and neurite outgrowth of cortical neuronunder oxidative stressObjective:The aim is to study the effect and the potential mechanism of Shh on neurite outgrowthin primary cortical neurons under oxidative stressMethods:Cortical neurons were obtained from the embryonic brains at E15-18 of C57BL/6 mice. Cells were pre-treated with 100 mM H2O2 for 2h, subsequently with the treatment of Shh(5/50ng/ml) for 24 h. Control experiments were performed with no Shh or H2O2 treatment, and the H2O2-injury group was treated with 100 mM H2O2 for 2 honly. The viability of cortical neurons was evaluated by Cell Counting Kit-8(CCK-8) assay. Immunohistochemistry andWestern blot were used to analyse the expression of GAP-43.Intracellular level of ROS was quantified by reactive oxygen species assay kit. Superoxide dismutase(SOD) activity was measured according to the instruction of SOD assay kit. Neuronal Malondialdehyde(MDA) levels were determined by Wasowiczs’ s method. JC-1 kit was employed to measure the mitochondrial membrane potential of cortical neurons. The cellular ATP levels were determined according to the protocol of ATP assay kit.The change of mitochondrial respiration complex II was examined with an ELISA kit.Results:1 H2O2 treatment decreased the cell viability by approximately 75% compared with the untreated control, and the decrease in cell viability was signi?cantly rescued by Shh(P<0.05). However, Shh at 50 or 500 ng/ml had a similar effect on cell viability.2 Neurons exposed to H2O2 showed a signi?cantly decrease in neurite outgrowth. In the presence of Shh for 24 hr after H2O2, on the other hand, the decrease in neurite outgrowth signi?cantly recovered(P<0.05). GAP-43 was markedly reduced after H2O2 without any treatment, while expression of GAP-43 was remarkably recovered by Shh treatment(P<0.05), suggesting that Shh could effectively restored neuritogenesis ability of neurons insulted by oxidative stress.3 Shh protects neurons by increasing ROS and SOD activity and decreasing the production of MDA under oxidative stress conditions(P<0.05).4 The normal structure of the neurons contained abundant mitochondria organelles and the intact appearance of the mitochondrial organelles. After expoured to H2O2, the mitochondria of the neurons showed severe damage. Swollen and fractured structures were observed. By comparison, the motichondrial morphology of the neurons in the Shh-treatment groups showed varying degrees of recovery, displaying a few swollen mitochondria and slightly broken mitochondria organelles.In addition, Shh attenuated the loss of MMP induced by H2O2 in cortical neuron cells(P<0.05).5 Shh significantly elevated the level of cellular ATP, which was reduceded by exposure to H2O2(P<0.05). 6. Shh significantly elevated the level of mitochondrial electron transport chain copmlex II, which was reduced by exposure to H2O2.Conclusions: We provided evidence that Shhhave neuroprotective and neurith outgrowth of cortical neuron against the detrimental efects of oxidative stress in the primary cultured corital neuron. Shh has been shown to potentlycounteract ROS release and prevent mitochondrial dysfunction as well as ATP and mitochondrial respiratory chain complex enzymes II activitiesagainst oxidative stress. Shh might be considered as a potential for therapeutic interventions that target mitochondria. |
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