| Objective: As the severe complication of subarachnoid hemorrhage (SAH), delayed cerebral vasospasm (DCVS) is responsible for the disability and mortality of patients with SAH. However, it isn't understood about mechanism of DCVS development, which couldn't find satisfied treatment. So it is very significant to investigate the mechanism further to improve the prognosis of patients with SAH. It has been investigated that it is a complicated multifactorial pathological process of vasospasm and the mechanism of smooth muscle contraction mediated by protein kinase C (PKC) plays important role in the pathogenesy of DCVS. PKC is a phospholipid dependent protein kinase activated by calciumion and an important factor of signal transduction. It is activated persistently and causes sustained contraction of vascular smooth muscle in SAH. It has been demonstrated that PKC inhibitor can relieve cerebral vasospasm in animal extraorgan experiment and recent viviperception. It is one of hot points to search PKC inhibitor in the DCVS study. In our study, we observed morphological changes of basilar artery in different phases and expression of active PKC determined by immunohistochemical staining and substrate phosphorylation methods by a rabbit SAH model. The aim is to assess preventive and therapeutic effect of breviscapine on DCVS after SAH and investigate its mechanism, which will provide theoretical principle for treating DCVS by breviscapine in the clinical application. Material and methods 1 fifty-four chinese rabbits were assigned randomly to three groups: ①SAH+saline group (n=18): We used two-hemorrhage model by injection of autologous aterial blood into cisterna magna on day0 and day2, and we injected 10ml saline slowly twice a day from 30 minutes after the first autologous aterial blood injection until animals were killed; ②SAH+breviscapine group(n=18): We built the same model and injected 4 milligram breviscapine per kilogram of animals twice a day from 30 minutes after the first autologous aterial blood injection until animals were killed; ③shamed operation group (n=18): Animals were anesthetized without puncture and injection. 2 Rabbits were deeply anesthetized and killed, brain stem and tissues with full-length basilar artery were taken from the skull and stored in iced phosphate buffered solution. The whole basilar artery were disassociated under the operating microscope, and two third of it were set in the liquid nitrogen for two minutes then were stored in the chill box. One third of basilar artery were fixed in 4% paraformaldehyde for 24 hours, then embeded in paraffin wax for histology and immunohistochemistry.3 The activity of protein kinase C was determined by immunohistochemistry and substrate phosphorylation: apply immunohistochemistry automatic analytic system Version 2.0 to record the percentage of the positive area of basilar artery and apply nonradioactive isotope determining PKC activity kit in substrate phosphorylation to calculate the activity of PKC. The unit of activity was signed as pmol/μg·min-1. 4 All the data were analyzed by Software Packaged by Social Science ver 12.0 and were expressed by mean ±standard deviation. T test was used for comparison of two groups and one-way ANOVA was used to compare the difference of three groups while SNK-q test for each two groups. The difference was considered statistically significant (P<0.05). Results 1 Hematoxin Eosin (HE) results: The structure of basilar artery is normal in shamed operation group. In SAH+saline group, there were changes in basilar artery including tunica externa and media thickening, corrugation of the tunica elastica interna, inflammatory infiltration and extracellular matrix increment on day4. While all the changes were more severe on day7 and less worse on day10. Compared with SAH+saline group, all the pathological changes were abated obviously on day4, day7 and day10 in SAH+breviscapine group. There was significant difference of the thickness of artery wall and inside diameter of basilar artery between SAH+saline group and shamed...
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