Effects Of The Artificial Synthetic E-selectin On The Expression Of NF-κB And MCP-1 In An Elastase-induced Model Of Common Carotid Artery Aneurysm In Rabbits

Partâ… Creating aneurysm model of rabbits common carotid artery by appling elastase and expression of NF-κB and MCP-1 in modelBackgrounds and objectives:An intracranial aneurysm (IA) is a cerebrovascular disorder in which weakness in the wall of a cerebral artery causes a localized dilation or ballooning of the blood vessel,a common location of cerebral aneurysms is on the arteries at the base of the brain,known as the circle of Willis.Intracranial aneurysm is the primary cause of subarachnoid hemorrhage (SAH).Its motality approaches 50% in the first month after rupture,aneurysm is a serious disease which endangers the life of the people.To provide an effective basis for etiological treatment of the aneurysm,studying of intracranial aneurysm pathogenesis is urgent and important.Researches on the aneurismal shape, pathogenesy and evolvement are very significant for clinical treatment,this all depend on making credible intracranial aneurysm model.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.In recent years,the research made on which inflammation and extracellular matrixenzyme system participate in the formation of aneurysm become more and more deeply.Numerous research of abdominal aortic aneurysm (AAA) indicated that infiltration of various kinds of inflammatory cells lead to impairment of elastic fibers and collagen fibers in the artery wall,thus the aneurysm happens. 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. We plan to use animal model to detect the early expression of NF-κB, MCP-1in the aneurysm wall, and discuss the possible mechanism of IA that can provide favorable experiment foundation for etiological treatment of IA.Methods:(1) 24 healthy New Zealand rabbits(2.5~3.0kg),half male and half female,were divided into 4 groups randomly,group A(control group),group B(one week),group C(two weeks),group D(three weeks),each group has 6 rabbits,feeding each rabbit in one cage freely in 20~25℃rooms.(2) 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 (CCA),avulse the superficial fibrous tissue of the point under the operation microscope,then drop 50u/ml elastase solution 0.2ml on adventitiaes of the point of the vessel,dropping PBS on control group,inject 500u heparin through rabbits posterior auricular vein 20 minutes later.(3) One week after the models created,use CT angiography (CTA) to detect the morphologic change of the right CCA and formation of the aneurysms in all rabbits.(4) From the operation day,execute 6 rabbits every week according to the animals groups,before executing,measure the maximum diameter of CCA in the dropping point,measure the neck width and height of the aneurysms at the same time, then execute the rabbits and collect the aneurysm samples.(5) Using HE dyeing and light microscope to examine the aneurysm samples.(6) Use immunohistochemistry to detect the expression of the protein of NF-κB and MCP-1.(7)Another freezing aneurysm samples of the first part were undertaken real-time polymerase chain reaction (PCR) that were used to detect the expression of the mRNA of NF-κB and MCP-1.Results:(1) CTA results and measurement:use CTA to detect all the 18 experimental rabbits in 7th day after operation,all of them have found visible fusiform or cystiform aneurysms where the elastase was dropped,and also found that some vessels were obstructed;aneurysm has not found in the control group,compared with group A and B respectively, the height and width of aneurysms of group C is larger(p<0.05),and there is no significant difference between group C and group D (p>0.05).(2) Pathological results: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.(3) Immunohistochemistry results:the immunohistochemistry results disclose that:compared with group A respectively,the NF-κB expression of group B,C and D are all more (p<0.05,<0.05,<0.05),and there is no significant difference between group B and group C (p>0.05).Compared with group A respectively,the MCP-1 expression of group B,C and D are all more (p<0.05,<0.05,<0.05), and there is no significant difference between group B and group C (p>0.05).The results indicate that NF-κB and MCP-1 all reach the peak at 1 week,and decreased at the 3rd week.(4) Real-time PCR results:the Real-time PCR detection consequence manifest that:compared with group A respectively,the NF-κB expression of group B,C and D are all more (p<0.05,<0.05,>0.05),and there is no significant difference between group B and group C (p>0.05). Compared with group A respectively,the MCP-1 expression of group B,C and D are all more (p<0.05,<0.05, >0.05), and there is no significant difference between group B and group C (p>0.05).Conclusions:(1) Dropping elastase on rabbits'CCA can create visible fusiform or cystiform aneurysms,it is a simple method with low cost and can be repeat easily.The pathological examination indicated that the aneurysm model has typical aneurysm morphological charateristics which is extremely similar to human's IA.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.(2) NF-κB activation plays a important 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 IA.(3) MCP-1 as an early express gene,its expression products induced macrophage sticking and infiltration that further degraded the elastic fibers in the aneurysmal walls,thus promoting the development and progression of IA. Partâ…¡Effects of the artificial synthetic E-selectin on the expression of NF-κB and MCP-1 in an elastase-induced model of common carotid artery aneurysm in rabbitsBackgrounds and objectives:IA is only rarely symptomatic unless they ruptured, which typically results in SAH,it is a kind of critical disease with high mortality and morbidity,and its incidence is increasing gradually.As the development of minimally invasive surgery and endovascular coiling technique,the morbidity and mortality of patients with ruptured aneurysm have decreased rapidly in recent years.However,the overall outcome of IA is still not perfect.Because,there is limited understanding about the biological mechanisms associated with the pathogenesis,growth,and rupture of intracranial aneurysms.So,we intend to use drugs to interfere with the formation of IA from the point of the inflammatory mechanisms, to prevent the occurrence of aneurysms,inhibit or delay the development of aneurysms,and prevent the aneurysms from rupturing. In recent years, the view of inflammatory response promoted the development of intracranial aneurysms is confirmed by many researchers at home and abroad,but,the research reports of anti-inflammatory treatment of aneurysms are rare. Based on the models of rabbits'common carotid artery aneurysms in the partâ… ,we use the artificial synthetic E-selectin interference aneurysms in order to observe its effect on on the expression of NF-κB and MCP-1 in an elastase-induced model of common carotid artery aneurysm in rabbits.Methods:(1)18 healthy New Zealand rabbits(2.5~3.0kg),half male and half female,were divided into 3 groups randomly,group E,F and G are treatment groups(E is the one week group,F is the two weeks group,G is the three weeks group);each group has 6 rabbits,feeding each rabbit in one cage freely in 20~25℃rooms.(2)Create the aneurysm models like as partâ… .(3) Use type 24GY intravenous detaining needles in right ear vein for all rabbits of experimental and treatment groups,then inject 1mg/(kgï¹'d) artificial synthetic E-selectin into right ear vein in treatment groups every day,until executed.(4)One week after the models created,use CTA to detect the morphologic change of the right CCA and formation of the aneurysms in all rabbits.(5)From the operation day,execute 6 rabbits every week according to the animals groups,before executing,measure the maximum diameter of CCA in the dropping point,measure the neck width and height of the aneurysms at the same time,then execute the rabbits and collect the aneurysm samples.(6)Using HE dyeing and light microscope to examine the aneurysm samples.(7)Use immunohistochemistry to detect the expression of the proteinum of NF-κB and MCP-1.(8)Another freezing aneurysm samples of the first part were undertaken real-time polymerase chain reaction (PCR) that were used to detect the expression of the mRNA of NF-κB and MCP-1.Results: (1)CTA results and measurement:use CTA to detect all the 18 experimental rabbits in 7th day after operation,all of them have found visible fusiform or cystiform aneurysms where the elastase was dropped,and also found that some vessels were obstructed;compared with group B,C and D respectively, the height and width of aneurysms of group E,F and G are all smaller(p>0.05,<0.05,<0.05),and there is no significant difference between group F and group G (p>0.05).(2)Pathological results:the pathological examination of the group E 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.,but the pathological changes in group F and G are better than group E. (3)Immunohistochemistry results:the immunohistochemistry results disclose that:Compared with group B,C and D respectively, the NF-κB expression of group E,F and G are less(p>0.05,<0.05,<0.05), and there is significant difference between group F and group G (p<0.05);Compared with group B,C and D respectively, the MCP-1 expression of group E,F and G are less(p>0.05,<0.05,<0.05), and there is significant difference between group F and group G (p<0.05).The results indicate that the artificial synthetic E-selectin can inhibite the expression of NF-κB and MCP-1 significantly after 2 weeks.(4) Real-time PCR results:the Real-time PCR detection consequence manifest that; Compared with group B,C and D respectively, the NF-κB expression of group E,F and G are less(p>0.05,<0.05,<0.05), and there is significant difference between group F and group G (p<0.05);Compared with group B,C and D respectively, the MCP-1 expression of group E,F and G are less(p>0.05,<0.05,<0.05), and there is significant difference between group F and group G (p<0.05).The results also indicate that the artificial synthetic E-selectin can inhibite the expression of NF-κB and MCP-1 significantly after 2 weeks.Conclusions:(1)The artificial synthetic E-selectin can delay or inhibit the reduction and atrophy of smooth muscle cells and breakage of elastic fiber breakage,thereby delaying or inhibiting the pathological process of aneurysms.(2)The artificial synthetic E-selectin can inhibit the expression of NF-κB and MCP-1 significantly after 2 weeks.It demonstrates that the anti-inflammatory treatment of the artificial synthetic E-selectin may delay or prevent further development of the aneurysm model.