| Objectives:Delayed cerebral vasospsam(DCVS) is a major cause of death and disability in patients with aneurysmal subarachnoid hemorrhage(SAH). It has been clear that expression of inflammatory cytokines was increased in the spastic artery after SAH and a critical event participating in such responses is the recruitment of circulating leukocytes into the inflammatory site. That inflammatory reaction of the cerebral artery may cause sustained contraction of the cerebral artery. Nuclear factor kappa B( NF-κB) , a main transcription factor of many inflammation medium and cell factor, is activated to lead to highly expression of TNF-a,ICAM-1 and IL-1.Our recent studies have demonstrated the expression of NF-κB in the development of delayed cerebral vasospasm after SAH in rabbits is high. But it was not clear whether there were changes of expression of TNF-a. In our study, We examined the expression of TNF-a and NF-κB in the development of delayed cerebral vasospasm by immunohistochemical and Western blot analysis in a rabbit SAH model. At the same time we apply cholecystokinin-8 ( CCK-8) by cisternal injection to prevent and cure DCVS, and discuss the molecular biological mechanism and testing its therapeutic effect on DCVS by observing action on the expression of NF-κB and TNF-a, and carried out a try to develop new drug. Methods:sixty-four chinese rabbits (weight range from 1.5kg to 2.0kg) were assigned randomly to four groups: ①shamed operation group(n=8):only shamed puncture and injecting blood;②SAH group (n=24). We used the two-hemorrage model of SAH ,and injecting autologous arterial blood into the cisterna magna on day0 and day2. Tweety-four animals in SAH group were killed respectively on day4, day7, and day14;③CCK-8 group(n=24): In CCK-8 group 0.5ml CCK-8 (the dosage is respectively 1ug/kg ,4ug/kg and 8ug/kg) were injected into the cisterna magna once every day,and animals were killed at day7;④saline group(n=8): same volume 37℃ physiological saline replace CCK-8 and and animals were killed at day7. Half of all animals were deeply anesthetized and perfused with Hanks balanced salt solution,ph7.4 at 37℃, 300ml then 2% paraformaldehyde in Hanks balanced salt solution,pH7.4,at 37℃,200ml was perfused . Perfusion was performed at a standard height of 100cm from the chest. The specimems stored overnight in 4% paraformaldehyde at room temperature were embeded in paraffin wax for Hematoxin eosin(HE) and immunohistochemistry. Under anaesthesia vertebrobasilar arteries of other animals was removed quickly and frozen right away at 70℃ below zero for Western blot analysis. Applying immunohistochemistry automatic analytic system Version 2.0 to record the percent of the positive cells and alphalmagerTM 1220 Documentation & Analysis system V5.50 to measure integral optical density (IOD) of TNF-a and NF-κB . The software package SPSS 10.0 was used for t test statistical analysis in control group and for F test between groups and P < 0.05 was considered statistically significant. Results: 1.Histological results: The structure of basilar artery is normal in shamed operation group(Fig1). The BA exhibited subendothelial thickening and severe corrugation of the tunica elastica interna in the SAH group at day4, day7 (Fig1). Compared with shamed operation group, diameter decreased and thickness increased in SAHday4 and day7 group(Table1P<0.01).It is significant severe vasospasm (Table1)especially in day7. Cerebral vasospasm in the CCK-8 group(Table2) was remarkably attenuated and related to dosage,compared with that in the SAHday7 group and the SAH+saline group . There is no differences between SAHday7 group and the saline group (Table2 P>0.05).2.Immunohistochemical: Little immunoreactivity of NF-κB and TNF-a can be found in shamed operation group (Table3 P>0.05). At day4 after injecting blood, The express of NF-κB and TNF-a began to increase(Table3), and it is most significant at day7 after injecting blood(Table3). Immunoreactivity decrease to level of control group at day1... |
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