The Pathogenesis Of Intracranial Aneurysm And The Interventional Effect Of Edaravone

Chapter one Regulatory mechanism ofTumor Necrosis Factor-alpha in intracranial aneurysmsObjectiveThe purpose of this study was to investigate the relationship between TNF-αpolymorphisms in intracranial aneurysms and explore the pathogenesis of aneurysms.MethodTo investigate the genetic variation of TNF-α expression in 192 intracranial aneurysm cases and 112 control groups of TNF-a-3959 T>C(rs1799964),4127 C>A(rs1800630),4133C>T(rs1799724),4184C>T(rs4248158)and 4752G>A(rs361525)were analyzed by polymerase chain reaction(PCR).Allelic frequency differences between patients and controls were examined.The differential expression of TNF-α in patients with intracranial aneurysm and normal controls was compared by reverse transcription-polymerase chain reaction(RT-PCR).The differences in elastin between the intracranial aneurysm wall and the control group(renal artery and superficial temporal artery)were compared by immunohistochemistry.The differences in the number and distribution of TNF-αpositive cells were analyzed in the intracranial aneurysm wall.ResultsThere was no statistical significance in the frequency of 4127C>A(P=0.072),4133C>T(P=0.373),4184C>T(P=0.749)and 4752G>A(P=0.184)genotypes in IA and control groups.However,The frequency of TNF-a-3959T>C allele variation was 90.6%in the intracranial aneurysm group and 0.9%in the control group.The T allele was closely related to the incidence of intracranial aneurysms in the Chinese population.There was significant difference in the expression of TNF-α between patients with intracranial aneurysm and normal RT-PCR controls.Compared with the normal control group by immunohistochemistry,the number of TNF-α positive cells in the aneurysm wall was significantly increased in patients with intracranial aneurysm.ConclusionThe frequency of TNF-a-3959T>C allele variation was was closely related to the incidence of intracranial aneurysms in the Chinese population.A new TNF-αlocuswas found to be closely related to the occurrence of intracranial aneurysms in Chinese population.The expression of TNF-α increased in patients with ruptured intracranial aneurysms compared with the control group.The distribution of TNF-α positive cells is very uneven.There are almost no positive cells seen at the thick wall of the intracranial aneurysms,and it is very dense at the weak wall of the intracranial aneurysms.This distribution is related to intracranial aneurysms bleeding.Chapter Two Effects of P22Phox-214TC Mutation on Intracranial AneurysmsObjectiveWe investigate the effect of the genetic variation of P22phox-214T/C on the expression of NADPH and its relationship with the occurrence of intracranial aneurysms.MethodOur experiment used polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)to analyze the polymorphism of P22phox-214T/C gene in 192 cases of IA and 112 normal controls.NADPH mRNA was analyzed by reverse transcription-polymerase chain reaction(RT-PCR).The experimental results of RT-PCR were verified by enzyme-linked immunosorbent assay(ELISA).Six IA patients were randomly selected to detect the expression of TNF-α,SOD,NADPH,and GAPDH.The difference in the number of P22phox positive cells in the control group of the intracranial aneurysm wall and the superficial temporal artery was compared by immunohistochemistry.ResultsIn clinical cases,the frequency of the P22phox-214C>T allele was 86.9%in the IA group,while that of the control group was 23.53%.The frequency of P22phox-214T/C allele variation in the intracranial aneurysm group was significantly different(P<0.001)compared to that of the control group.Consistent with the results of p22Phox-214T>C single nucleotide variation,the mRNA expression of NADPH in the intracranial aneurysm group was significantly higher than that of the control group.The NADPH levelin serum of the intracranial aneurysm group were significantly higher than thatof the control group(P<0.0001)by ELISA.After intracranial aneurysm hemorrhage,SOD and NADPH were significantly higher than TNF-α,and the expression of oxidative stress products increased more significantly than that of inflammation-related products.The number of P22phox positive cells in the aneurysm wall was similar in both the patients with intracranial aneurysm and those in the normal control group compared by immunohistochemistry.ConclusionP22phox-214C>T single nucleotide variation affects NADPH expression and intracranial aneurysm development,NADPH promotes the incidence of intracranial aneurysms.Oxidative stress also plays an important role in rupturing hemorrhage in IA patients.The expression of oxidative stress products is more pronounced than that of inflammation-related products.The effect of p22Phox on intracranial aneurysm is different from that of inflammatory factors,which may be influenced by the increase of NADPH in peripheral blood.Chapter Three Evaluation and Comparison of Three Elastase Induced Rabbit Common Carotid Arterial AneurysmsObjectiveWhite rabbits were often used to create clinical aneurysm model.This study analyzed and compared three methods of creating rabbit common carotid artery aneurysms,aiming at exploring a simple,safeand effective way to create elastase-induced aneurysms.Method42 Japanese white rabbits were treated with elastase ablation to create common carotid artery(RCCA)aneurysm models.42 male adult Japanese white rabbits(3.05±0.65 kg)were randomly divided into 3 groups:RCCA root medication group(group A,n=12),mid-RCCA medicationgroup(group B,n=18),and the ligated RCCAgroup(group C,n=12).For GroupA:the origin of the RCCAwas occluded with a temporary aneurysm clip,and a residual lumen wascreated by temporary aneurysm clip 2 cm away from the origin.The residual lumen was perfused with elastase for 20 minutes and then the clip was removed.For GroupB:the middle part of RCCA was treated the same way as GroupA.For GroupC:the middle the middle part of RCCA was treated asGroupB,and then the distally RCCA was ligated.Color Doppler ultrasound studies were then performed for blood flow measurements inside the aneurysm pouch after the aneurysm models created for 3 weeks.Prior to sacrificing animals,angiography studies were performed.Harvested aneurysmal tissues were used for histological analysis,SMA-a,CD31,CD34,CD68,collagen IV,Ki67 and the relevant indexes were compared with those of human intracranial aneurysms.ResultsCompared with human intracranial aneurysm specimens,all three methods successfully established an elastase-induced aneurysm model.Histology showed that biological responses were similar to both human cerebral aneurysms and previously published elastase-induced rabbit aneurysm models.GroupA and B had the same morphology,but Group A had a higher mortality rate than that of GroupB.GroupB and GroupC had different morphology.The aneurysm of GroupC was more similar to human cerebral aneurysms but had higher mortality rate than GroupB.GroupB confirmed an alternate method for creating elastase-induced aneurysms on the middle of RCCA and that this was a safe,effective method.ConclusionThrough analysis and comparison,the Scheme B is proved to be the simplest,safest,and most effective modeling method.The aneurysm model established by Scheme B can be used for basic research related to aneurysm mechanism.We have provided a new and effective method for basic research on aneurysm.Chapter Four The role of high mobility group box-1 in the development of intracranial aneurysmsObjectiveWe investigated the expression of high mobility group box-1(HMGB-1)in the ruptured human intracranial aneurysm wall and plasma,and verified the clinical experimental results in therabbit ear common carotid aneurysm model to explore the role of HMGB-1 in the development of intracranial aneurysms.MethodReverse transcription polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to analyze the expression of HMGB-1 in 192 cases of intracranial aneurysms and 112 controls.The expression of HMGB-1 was measured by RT-PCR and ELISA in the rabbit model of common carotid artery aneurysm.By analyzing and comparing the results of human intracranial aneurysm wall by immunohistochemistry,RT-PCR and ELISA results were verified and the correlation between them was deduced.ResultsThere was a significant difference in HMGB-1 between the intracranial aneurysm group and the control group(P<0.001).The detection of related indicators of rabbit models of common carotid artery aneurysms showed that animal models have similar results to human intracranial aneurysms.The HMGB-1 results of immunohistochemistry also showed a significant difference between the intracranial aneurysm group and the control group.ConclusionThe expression of HMGB-1 was unevenly distributed in the aneurysm wall and was highly consistent with the positive cell expression characteristics of TREM-1 and TNF-α.Several inflammatory mediators may act synergistically and affect the occurrence and rupture of aneurysms.HMGB-1 is associated with the rupture of intracranial aneurysms in Chinese population.HMGB-1 is an important factor in the development of intracranial aneurysms.HMGB-1 is expected to be a new therapeutic target for the prevention of intracranial aneurysmsrupture.Chapter Five Modulating the growth of fusiform aneurysm in the common carotid arteryofrabbitsby oxygen free radical scavenger edaravoneObjectiveWe further validated the role of P22phox in the development of intracranial aneurysms.We determined the efficacy of edaravone in the treatment of fusiform aneurysms in the common carotid artery of rabbits and determined the therapeutic effect of oxygen free radical scavenger edaravone.MethodWhite rabbit aneurysm model was established first.These white rabbits were randomly divided into three groups:edaravone group(Edaravone 2 mg/kg/d,n=18)and control group(normal saline,4 mL/d),n=18)and normal groups(normal rabbits,n=10).From day 7 to day 21 after the preparation of the aneurysm,edaravone or physiological saline was intravenously injected twice daily into ear-bearing white rabbits for 14 consecutive days.Immunohistochemical staining of BCL-2,CD31,CD34,CD68,Ki67 SMA,and type II collagen and macrophages was performed to understand the changes of inner membrane and composition of lesion cells.Using EDTA as an anticoagulant to collect the plasma of white rabbits and detect the content of the test factors in plasma by ELISA:specifically TNF-α,MMP-2,matrix metal Protease 9(MMP-9),3-nitrotyrosine(3-NT),reduced coenzyme II(NADPH),myeloid trigger receptor-1(TREM-1),and high mobility group box protein B1(HMGB-1)Superoxide Dismutase(SOD),Hydrogen Peroxide(H2O2).ResultsThe success rate of the edaravone group aneurysm model was 62.5%,which was lower than that of the control group 82.3%;the diameter of the aneurysm was smaller than the control group(3.26±0.13mm vs.3.85±0.07mm).In the animal aneurysm model,edaravone inhibited the development of aneurysms.The MMP-9 levels in the edaravone group serum were significantly lower than those in the control group(P<0.05),but there was no significant statistical significanceof MMP-2 level between the edaravone group and the control group(P=0.7544).The serum levels of HMGB-1and TREM-1 in the edaravone group were significantly lower than those in the control group(P<0.05),and there were no significant differences in other parameters.After treatment with edaravone,there were no significant differences in H2O2 and SOD between aneurysm control group and normal control group.There were significant differences between 3-NT and NADPH edaravone treatment group and normal control group,but with There was no significant difference in the aneurysm control group.ConclusionEdaravone regulate the incidence of intracranial aneurysms is not mainly by regulating the production of oxidative stress-related substances,but mainly by affecting the expression of inflammation-related factors to affect intracranial aneurysms.Oxidative stress-related products and inflammation-related factors have a synergistic effect on the production of IA.Edaravone can inhibit the growth of aneurysms by regulating inflammation-related factors such as TNF-α,HMGB-1 and TREM-1.This study provideshelpful support and direction for the development of drugs for the treatment of intracranial aneurysms in the future.