| Objective: Cerebral ischemia is the first leading cause of death and the most frequent cause of permanent disability in adults in our country. Although the neurons can not regenerate, but clinical data show that survived stroke patients are showing varying degrees of functional recovery, suggesting that the brain has the ability to repair itself. In recent years, a large body of evidence showed that neural plasticity played an important role in functional recovery of stroke patients and animal models of cerebral infarction. At structural level, neural plasticity could be defined in terms of dendritic and axonal arborization, spine density, synapse number and size, receptor density, and in some brain regions also the number of neurons. These structures constitute the neural network and functional recovery after stroke foundation. Therefore, how to improve neural plasticity in the cerebral infarction provides new therapeutic strategies for the clinical treatment.Sonic hedgehog(Shh) signaling pathway is a highly conserved secreted glycoprotein. Shh is one of the family of the hedgehog protein which includes Sonic hedgehog(Shh), Desert hedgehog(Dhh) and Indian hedgehog(Ihh). The binding of Shh protein with Ptch will induce pro-survival response of Gli family within the cell-mediated. The Gli family of vertebrates includes Gli1, Gli2 and Gli3. Once the Gli family of transcription factors are activated, they can translocate from the cytoplasm to the nucleus. Studies have shown that Shh signaling pathway is essential in the embryonic of central nervous system during development. which can regulate cell differentiation and proliferation. Simultaneously, it can induce regeneration of neural stem cells after brain injury, and participate in vascular remodeling. Our preliminary results showed that Shh signaling pathway would reactivate after cerebral infarction. The expression of Shh /Ptch1/Gli1 in cortical ischemic tissue was significantly increased compared with the control group. When Shh/Ptch1/Gli1 signaling pathway was specifically blocked, the infarct volume of brain damage will increase. Based on the above data, we hypothesized that Shh signaling pathway is neuroprotective in stroke.Growth associated protein 43(GAP43) is parts of axons and presynaptic, which can be expressed at high levels in the process of developing axons. GAP43 can be used as a reliable marker of new axonal growth in brain development, brain injury or cerebral ischemic process. The protein of brain-derived neurotrophic factor(BDNF) is widely distributed in the central nervous system. BDNF plays an important role in central nervous system during development, neuronal differentiation, growth and survival. Besides, it can prevent the death of injured neurons, improve the pathological state of neurons and promote the regeneration and differentiation of injured neuronal. Thus, BDNF is crucial in mature neurons of the central and peripheral nervous system, which can maintain the survival and normal physiological functions of neurons.Dl-3-n-butylphthalide(NBP), also known as NBP, is a kind of ourself-developed drugs which can treat ischemic cerebrovascular disease. NBP was originally extracted from the celery seed. Previous studies have shown that NBP had a favorable effect in cerebral ischemia. It can significantly improve the microcirculation of cerebral ischemia and blood flow. It can also increase the number of capillaries in ischemic area and reduce infarct size and mitigate cerebral edema. NBP can inhibit neuronal apoptosis, and improve energy metabolism. And it can also suppress platelet aggregation and thrombosis. However, the mechanism related to NBP in neurological recovery after stroke is still unclear. In present study, we used the model of distal middle cerebral artery occlusion(d MCAO) in C57BL/6 mice and observed the neuroprotective of NBP from acute post-operative period to 4 weeks after cerebral ischemic injury. And we observed NBP’s effect on Shh/Ptch1/Gli1 pathway, as well as the expression of GAP43 and BDNF. Moreover, we explored the impact of NBP on axons, dendrites, and dendritic spines.Methods: This study was consisted of three sections: Experiment 1 was used to detect NBP’s neuroprotective effect in focal cerebral ischemia. Experiment 2 was conducted to evaluate NBP’s effect on the expression Shh/Ptch1/Gli1, GAP43 and BDNF. Experiment 3 was used to detect the NBP’s effect on cortical tissue axonl sprouting, dendrites and dendritic spines outgrowing.Experiment 1, seventy-two male C57BL/6 mice were divided randomly and equally into acute phase parts and chronic phase parts after d MCAO and each group contained three groups(12 mice in each group): Vehicle group(Vehicle); Low dose group(NBP-L) and High dose group(NBP-H). NBP-L group and NBP-H group were prepared in d MCAO model and immediately administered NBP(20 mg/kg or 40 mg/kg) after 1 hour in the abdominal. Vehicle group were also operated in d MCAO model and injected with Tween-80(0.5%) in the same way with NBP groups. Neurological deficit was evaluated by a rotarod test in acute phase at 3 days, 4 days, 5 days, 7 days, 8 days, 9 days, 14 days, 15 days and 16 days after d MCAO, while in chronic phase it was tested at 14 days, 15 days, 16 days, 28 days, 29 days, 30 days after d MCAO.Experiment 2, 48 mice were randomly and equally divided into four groups:(12 mice in each group): Sham operated group(Sham); Vehicle group(Vehicle); Low dose group(NBP-L) and High dose group(NBP-H). NBP solution(20 mg/kg or 40mg/kg) was administrated through intraperitoneal injection. Immunohistochemistry staining and confocol technique staining were used to detect the location of GAP43. Western blotting was used to analyse the expression of Shh/Ptch1/Gli1, GAP43 and BDNF.Experiment 3, 18 mice were randomly and equally divided into two groups(9 mice in each group): Vehicle group(Vehicle) and Low dose group(NBP-L). NBP solution(20 mg/kg) was administrated through intraperitoneal injection. Neurons anterograde tracer(Biotin dextran amine, BDA) was utilized to detect cortical tissue axonl sprouting. Golgi stain was used to investigate the effects of NBP on dendrites and dendritic spines outgrowing.Results:1 LSD test analysis of one-way ANOVA was conducted to detect the neurologic deficit scores by rotarod test in acute phase and chronic phase after d MCAO. Mice in Vehicle group, NBP-L group, NBP-H group performed a left palsy. In acute phase, compared with Vehicle group, the scores in NBP-L group were increased at 4 days, 5 days, 7 days, 8 days, 9 days, 14 days, 15 days and 16 days(Vehicle vs. NBP-L: 4 days: 38.37±3.53 vs. 51.14±3.31, P < 0.05; 5 days: 43.74±3.34 vs. 56.67±3.96, P < 0.05; 7 days: 44.48±2.69 vs. 66.16±4.39, P < 0.05; 8 days: 44.42±2.28 vs. 63.41±3.68, P < 0.05; 9 days: 39.24±2.95 vs. 69.94±5.14, P < 0.05; 14 days: 38.97±4.33 vs. 57.24±3.92, P < 0.05; 15 days: 36.25±4.16 vs. 50.62±4.28, P < 0.05; 16 days: 33.19±5.27 vs. 51.14±5.22, P < 0.05). Compared with Vehicle group, the scores in NBP-H group were incresesd at 4 days, 5 days, 7 days, 9 days and 14 days(Vehicle vs. NBP-H: 4 days: 38.37±3.53 vs. 48.51±3.52, P < 0.05; 5 days: 43.74±3.34 vs. 55.32±3.93, P < 0.05; 7 days: 44.48±2.69 vs. 66.37±4.87, P < 0.05; 9 days: 39.24±2.95 vs. 69.94±5.14, P < 0.05; 14 days: 38.97±4.33 vs. 56.73±4.93, P < 0.05). In chronic phase, compared with Vehicle group, the scores in NBP-L group were incresed at 14 days, 15 days, 16 days, 28 days, 29 days and 30 days(Vehicle vs. NBP-L:14 days: 22.30±1.55 vs. 31.45±2.34, P < 0.05; 15 days: 31.42±2.07 vs. 42.28±2.56, P < 0.05; 16 days: 28.88±2.28 vs. 39.11±2.30, P < 0.05; 28 days: 31.24±2.65 vs. 41.61±2.37, P < 0.05; 29 days: 31.66±2.73 vs. 42.60±2.57, P < 0.05; 30 days: 31.94±2.89 vs. 52.70±3.29, P < 0.05). However no statistical difference was observed between vehicle group and NBP-H group in chronic phase.(n = 12 in each group).2 GAP43 was showed in immunohistochemistry staining and confocol technique staining in the cerebral cortex after d MCAO.3 NBP’s role in the expression of Shh/Ptch1/Gli1, GAP43 and BDNF. We analyzed the expression of Shh/Ptch1/Gli1, GAP43 and BDNF and nuclear Gli1 at protein level after treating with NBP-L and NBP-H in ischemia brain by Western blot. Compared with Vehicle group, NBP-L treatment showed upregulated the expression of Gli1 at 3 days and 7 days after d MCAO. Compared with Vehicle group, NBP-L treatment upregulated the expression of Ptch1 at 7days, 14 days at protein levels(P < 0.05). The expression of Shh at 14 days and the expression of BDNF at 3 days and 14 days are also upregulated in NBP-L group after compared with Vehicle group at protein levels(P < 0.05). Compared with Vehicle group, NBP-H treatment upregulated the expression of Ptch1 at 14 days at protein levels(P < 0.05). The expression of BDNF at 3 days and 14 days are also upregulated in NBP-H group after compared with Vehicle group at protein levels(P < 0.05). However, the expression of GAP43 was no significant difference among Sham,Vehicle group, NBP-L and NBP-H groups(P > 0.05).Compared with Vehicle group, NBP-L and NBP-H treatment upregulated the expression of Gli1 at 14 days, 28 days at nucleus protein levels.4 In Golgi staining, there was a significant change in morphology and the number of dendrites as well as dendritic spines in NBP-L group, compared with the Vehicle group. In BDA tracer staining, there was significant changes in axonal regeneration in NBP-L group, compared with the Vehicle group.Conclusions: Above all, our findings showed that systemic administration of NBP improved the neurological deficits. The up-regulation of Shh/Ptch1/Gli1 may be involved in the potential neuroprotection of NBP. Moreover, NBP may possess neuroplasticity protective activey in cortex, based on the Golgi staining and BDA tracer staining. Thus, we concluded that NBP may represent a potentially beneficial therapeutic avenue for focal cerebral ischemic stroke treatment. |
PDF Full Text Request