Experimental Study On The Cerebral Aneurysm Model In Rabbits And The Expression Of NF-κB, MCP-1 And MMP-9 In The Model

PartⅠCREATING ANEURYSM MODEL OF RABBIT INTERNAL CAROTID ARTERY BY APPLING ELASTASEBackgrounds and purpose: Intracranial aneurysm (An) is the primary cause of subarachnoid hemorrhage (SAH), its lethiferous and disable ratio are very high. Although a great quantity of epidemiology have found that, many factors just like high blood pressure, diabetes, smoking, drinking and so on, are the risk factor of intracranial aneurysm, the development and breaking mechanism of the aneurysm have not been clearly illuminated. Researches on the aneurismal shape, pathogenesy and evolvement are very significant for clinical treatment, this all depend on making credible intracranial aneurysm model. At present, there are about five main methods of building aneurysm model, which are vein transplantation, artery transplantation, artery structure destruction, change blood hemodynamics and blood vessel intervention, but these models have some distance from ideal model. Nowadays there are more and more research on elastase-induced aneurysm models, which is simple and repeatable easily with low cost. The studies of pathology have indicated that elastase-induced aneurysm model is almost consistent with human's cerebral aneurysm and more closed to the ideal aneurysm model than models created by other methods. But there are some deficiencies about the morphologic and pathological changes of early period of elastase-induced aneurysm model, the study on morphologic observation and pathological changes have more significance which can further confirm the reliability of the aneurysm model, it also can establish foundation of further study on the pathogenesis and therapeutic measure of aneurysm.Methods: 24 healthy New Zealand rabbits, half female and half male, were randomly divided into 4 groups: group A(control group); group B(one week); group C(two weeks); group D(three weeks), 6 rabbits per-group. Measure the diameter of near heart point of the vessel which about 0.5mm~1.0mm far from the bifurcation of right common carotid artery. Avulse the superficial fibrous tissue of the point under the operation microscope and then drop 50u/ml elastase solution 0.2ml on adventitiaes of the point of the vessel. Dropping PBS on control group. Twenty minutes later, inject 500u heparin through rabbits posterior auricular vein. After a week, use CT angiography to detect the morphologic change of the right common carotid artery and formation of the aneurysms in all rabbits of control groups. From the operation day, execute 6 rabbits every week. Before executing, measure the maximum diameter of common carotid artery in the dropping point, meanwhile measure the neck width and height of the aneurysms. Then execut and collect the aneurysm samples, which are used to HE dyeing, immunity histochemical stain and Real-time PCR detection. Using HE dyeing and light microscope to examine the aneurysm samples.Results: After a week, use CT angiography to detect all the 18 experimental rabbits, all of them have found visible fusiform or cystiform aneurysms where the elastase was dropped, and also found that some vessels were obstructed. Aneurysms have not found in all the control group rabbits. The pathological examination of the experimental aneurysms showed that the wall of aneurysm becoming thinner, the internal elastic layers were disrupted or disappeared and the reduced medial vascular smooth muscle cell becoming atrophy or degradation obviously. Inflammatory cell infiltrate were also observed in the control group, but the internal elastic layers degradation and vascular smooth muscle degeneration were not found.Conclusion: Dropping elastase on rabbit common carotid artery can induced visible fusiform or cystiform aneurysms, this method is simple and repeatable easily with low cost. The pathological examination indicated that the aneurysm model has typical aneurysm morphological charateristics which is extremely similar to human's cerebral aneurysm. The elastase-induced aneurysm model can be used to study the etiology and treatment of aneurysm, it can also provide experimental platform for further study on the aneurysm.PartⅡEXPRESSION OF NF-κB, MCP-1 AND MMP-9 IN EXPERIMENTAL INTERNAL CAROTID ANEURYSM RABBIT MODELBackgrounds and purpose: Cerebral aneurysm (CA) is a common cerebrovascular disease (CVD), and the incidence is increasing gradually. Although more and more scholars have payed close attention to the disease, the mechanisms of the initiation and progression of CA remain to be elucidated. Recent years, the research made on which inflammation and extracellular matrixenzyme system participate in the formation of aneurysm become more and more deeply. A great quantity of exterior and interior investigations strongly suggests that chronic inflammation and reconstitution of the extracellular matrix in the vascular wall may be the main mechanism of the initiation and progression of the aneurysm. Numerous research of abdominal aortic aneurysm (AAA) indicated that infiltration of various kinds of inflammatory cells promote the secretion of matrix metalloprotease (MMPs), especially the MMP-9 secretion, so that lead to impairment of elastic fibers and collagen fibers in the artery wall, thus the aneurysm happens. The standpoint widely accepted by exterior and interior scholars. In the infiltrative inflammatory cells, the effect of macrophage is most outstanding. Monocyte chemotactic protein-1 (MCP-1) plays a crucial role in macrophage infiltration, and NF-κB regulated the expression of proinflammatory genes such as MCP-1 in the transcriptional level. But now there is so little correlated research between NF-κB, MCP-1, MMP-9 and the inflammatory mechanism of initiation and progression of CA that it needs further study. We plan to use animal model to detect the early expression of NF-κB, MCP-1, MMP-9 in the aneurysm wall, meanwhile, combine the present condition to discuss the possible mechanism of CA that can provide favorable experiment foundation for further study of CA.Methods: Take six aneurysm samples every group of paraffin blocks from the first part experimental and normal control group, then made into sectserial sections, use immunohistochemistry to detect the expression of the proteinum of NF-κB, MCP-1 and MMP9. Another freezing aneurysm samples of the first part were undertaken real-time rolymerase chain reaction(PCR)that were used to detect the expression of the mRNA of the three indicatrixes.Results:①The immunohistochemistry result disclose that: compared with the NF-κB control group, the one week group: p<0.001, two week group: p<0.001, and three week group: p=0.348; among which, the difference has no significance between the one week group and two week group (p=0. 516).Compared with the MCP-1 control group, the one week group: p<0.05, two week group: p<0.05, and three week group: p<0.05; among which, the difference has no significance between the one week group and two week group (p=0. 367).Expression of MMP-9 has increased gradually, the difference all have significance between every group (p<0.05).The result indicate that NF-κB and MCP-1 all reach the peak at 1 week,and decreased at 3 week. MMP-9 was increased gradually. These all located at smooth muscle cell, endothelial cell and inflammatory cell.②The Real-time PCR detection consequence manifest that: compared with the NF-κB control group, the one week group: p=0.041, two week group: p=0.008, and three week group: p=0.558; among which, the difference has no significance between the one week group and two week group (p=0.317).Compared with the MCP-1 control group, the one week group: p=0.001, two week group: p=0.006, and three week group: p=0.056; among which, the difference has no significance between the one week group and two week group (p=0. 124).Expression of MMP-9 has increased gradually, the difference all have significance between every group (p<0.05). Conclusion:①NF-κB activation plays a crucial role in the inflammatory reaction and pathological change of the cerebral aneurysm wall, so it may be one of the initiating agents in the initiation and deveiopment of the cerebral aneurysm.②MCP-1 gene as an early express gene, its expression product induced macrophage sticking and infiltration that further degraded the elastic fibers in the aneurysmal walls, thus promoting the development and progression of CA.③Destruction of the elastic fibers is one of the key factors in cerebral aneurysm (CA) formation. However, inflammatory cells infiltration and more MMP-9 secretion are the major cause of the elastic fibers destruction.