Influences Of Inhibiting Sonic Hedgehog Signaling Pathway On Vascular Regeneration In Rats With Acute Cerebral Ischemia

Background and ObjectiveWith the faster social rhythm, the changing of diet and lacking of physical activities and other factors, the incidence of cerebrovascular disease is increasing year by year, and most of them are young people. The incidence of ischemia cerebrovascular disease (ICVD) was in the first place in all cerebrovascular disease, which makes ICVD became one of the most common and fatal disease. Studies have found that prognosis of the ICVD patients with higher density of blood vessels is much better than the lower one, and Sonic Hedgehog (Shh) signaling pathway may be involved in the vascular regeneration after acute cerebral ischemia.Shh signaling pathway is mainly composed by signal peptide (Shh, Ihh, Dhh), transmembrane receptors (Ptch, Smo) and downstream transcription factor (Gli protein family), GLI1protein family plays a very important role in the activation of Shh pathway. Recent studies found that Shh signaling pathway in ischemic brain tissue after cerebral infarction was activated. Exogenous Shh can promote vascular regeneration and reconstruction after cerebral infarction and increase blood perfusion in the ischemic tissues and promote the restoration of neurological function. But, the mechanism is still unknown. In order to explore the mechanism, permanent middle cerebral artery occlusion models were produced for next experiment. We investigated the expression of Shh signaling pathway downstream factor Gli1、 Angiogenesis related factors, such as VEGF、bFGF and Angiogenesis markers CD105in rats with cerebral infarction through the use of cyclopamin, one antagonist of Shh signaling pathway, to explore the stimulative mechanism of Shh signaling pathway on vascular regeneration after cerebral infarction in rats.Materials and MethodsPermanent middle cerebral artery occlusion models were produced by suture emboli technique in rats.162healthy male SD rats, weight280-330g, were selected and divided randomly into three groups:sham operation group, infarction group and cyclopamine treatment group. Each group was divided into three subgroups (6h,24h,48h after Cerebral infarction). In cyclopamine treatment group, cyclopamine was given immediately after PMCAO by0.18mg/kg abdominally, while the same dose ethanol was given in cerebral ischemia group and sham operation group. Each subgroup took out8rats which were anesthetized and quickly took out the tissue of the ischemic brain cortex and stored in liquid nitrogen for the investigation of the expression of GLI1mRNA at different time points. The remaining10rats were perfused and fixated through the heart, then the tissue of the ischemic brain cortex were removed quickly to make paraffin section. The expression of Gli1mRNA in ischemic brain cortex was investigated by RT-PCR,and the expression of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (bFGF) and CD105was investigated by immunohistochemistry.Results(1) The determination results of Glil mRNA in ischemic brain cortex The expression of Gli1mRNA in model group and cyclopamine treatment group were began to rise at6hour,reached the peak at24hour, and began to decrease at48hour. The expression of Glil mRNA in model group were increased compared with sham operated group, and the difference have statistical significance. The expression of Gli1mRNA in cyclopamine treatment group was decreased significantly than in the model group, and the difference is statistically significant.(2) The determination results of VEGF in ischemic brain cortex From the results of Immunohistochemical staining, we found tan particles in the cytoplasm, which is the positive expression of VEGF. VEGF was expressed in neural cells, vascular endothelial cells and glial cells. The expression of VEGF in model group was increased significantly compared with sham operated group, and it reached the peak at24hour, began to decrease at48hour, and the difference between them have statistical significance(P<0.05). The expression of VEGF in cyclopamine treatment group was less than the model group (P<0.05).(3) The determination results of bFGF in ischemic brain cortex From the results of Immunohistochemical staining, we found tan particles in the cytoplasm, which is the positive expression of bFGF. bFGF was expressed in neural cells,vascular endothelial cells and glial cells, but was mainly in the neural cells. We could see a small amount of bFGF in sham operation group. The expression of bFGF in model group was increased significantly compared with sham operation group, and it reached the peak at24hour, began to decrease at48hour. The difference have statistical significance (P<0.05). The expression of bFGF in cyclopamine treatment group was less than the model group (P<0.05).(4) The determination results of CD105in ischemic brain cortex From the results of Immunohistochemical staining, we found tan particles in the cytoplasm, which is the positive expression of CD105. CD105was mainly expressed in vascular endothelial cells, and we could see trace amounts of CD105in sham operation group. The expression of CD105in model group was increased significantly compared with sham operation group(P<0.05), and it was continue to increase in a certain period of time. The expression of CD105in cyclopamine treatment group was less than the model group, and the difference have statistical significance(P<0.05).Conclusion(1) The expression of the signaling pathway downstream effectors Gli1was reduced by cyclopamine, an antagonist of Shh signaling pathway.(2) Shh signaling pathway is closely related to the vascular regeneration after cerebral ischemia. Inhibiting Shh signaling pathway can reduce the expression of VEGF, bFGF, CD105, and the regeneration of vascular as well.