Basal And Clinical Research On Subarachnoid Hemorrhage

Subarachnoid hemorrhage(SAH) is that the blood flows over to the surface of the encephalon and into the lacouna of the cerebrospinal fluid,which is divided into two classes:traumatic and spontaneous in clinic,the latter is divided into two classes: primary and tardive.Tardive subarchnoid hemorrhage is that the entorrhagia of the parenchyma of the encephalon perforates the brain tissue into subarchnoid hemorrhage;primary subarchnoid hemorrhage is that the angiorrhexis of the encephalon in the basalar portion and the surface and that the blood flows into subarchnoid hemorrhage in all reasons.In general,so-called subarchnoid hemorrhage, traumatic or spontaneous subarchnoid hemorrhage scilicet primary subarchnoid hemorrhage,which the etiopathogenisis is because the intracranial aneurysm ruptures in 85%,which forms aneurismal subarchnoid hemorrhage,perimesencephalic nonaneurysmal subarachnoid hemorrhage in 10%,other rare subarchnoid hemorrhage in 5%.After subarchnoid hemorrhage,the bulk of patients have been suffered cerebral vasospasm,which causes serious ischemic or delay ischemic brain damage in the region of the brain,further more,leading to cerebral infarction,causing death and mutilation,but the pathogenesy of subarchnoid hemorrhage and cerebral vasospasm is not clear.Our studies are aim at approaching the pathogenesy of subarchnoid hemorrhage and cerebral vasospasm from the point of view of molecular biology and clinic,so that enacting valid and effective therapeutic regimen and improving the prognosis of patients in subarchnoid hemorrhage.Part 1 Risk of Cerebral Vasospasrn in Subarachnoid Hemorrhage is Associated with Endothelial Nitric Oxide Synthase Gene PolyrnnorphismObjective:To study whether polymorphisms in the endothelial nitric oxide synthase gene(eNOS) may be implicated in the development of cerebral vasospasm in subarachnoid hemorrhage.Method:1.Three groups of subarachnoid hemorrhage patients were selected to study this hypothesis:cases with cerebral vasospasm in aneurysmal subarachnoid hemorrhage(ASAH)(N=98),cases with cerebral vasospasm in traumatic subarachnoid hemorrhage(TSAH)(N=96),and without cerebral vasospasm in aneurysmal or traumatic subarachnoid hemorrhage(N=195).Parents of 194 cases and 100 controls were also examined and were used for the transmission disequilibrium test,a family-based study design to test association.2.All patients are detected as follow:contain 3D-DSA,CT and TCCD.Transcranial color duplex sonography(TCCD) detection:the imaging of the vascellum in the outside of cranium is formed by using the detecting head of pulse in 4～12MHZ,of intracalvarium in 2～4MHZ.The detecting head of TCCD being put on the segment inferior of cervical part,on the anterior border of sternomastoid muscle,on anodic of collarbone,or through temple window or pulvinar window detects the PSV,MVF and EDV in the ECA,ICA,ACA,MCA,PCA,BA and VA,respectively and records the PSV,MVF and EDV respectively.The patients of the three groups been detected once all in the third day and the seventh day differently of the posthemorrhagic.3.The DNA of the peripheral blood of patients been extracted,by using two pair primers,which determining the 223-bp fragment containing the T-786C polymorphism,the upstream primer is 5'-GCATGCACTCTGGCCTGAAGTG-3', the downstream primer is 5'-CAGGAAGCTGCCTTCCAGTGC-3',and which determining the 267-bp fragment containing the G894T substitution in exon 7,the upstream primer is 5'CTGGAGATGAAGGCAGGAGAC-3',the downstream primer is 5'-CTCCATCCCACCCAGTCAATC-3'.Amplified by using PCR:Polymerase chain reaction(PCR) was carried out in a 50μL volume with 80 ng of genomic DNA, 50 pmol of each primer,200μmol/L dNTP,1.25 units of displayTAQ FL DNA Polymerase(PGC Scientific Co.,Frederick,MD,USA) with 5μL of 10×PCR buffer containing 15 mmol/L MgCl2(PGC Scientific Co.).Initial denaturation was performed at 94℃for five minutes and was followed by 40 cycles consisting of 94℃for one minute,55℃for one minute,and 72℃for one minute.Amplification was finished with extension at 72℃for five minutes.Bloting after amplifying:Eight microliters of the PCR-amplified product were added to 50μL of denaturing solution [500 mmol/L NaOH,2.0 mol/L NaC1,25 mmol/L ethylenediaminetetraacetic acid (EDTA),0.0001%bromophenol blue]and transferred onto a nylon membrane placed on a dot-blot apparatus under vacuum.After transfer,the membrane was immersed for two minutes in a 2×sodium chloride/sodium citrate solution and was dried for 20 minutes at 80℃.To determine the T-786C polymorphism,two 17 bp ASO probes were used with the following sequences:5'-CTGGCCGGCTGACCCTG-3' and 5'CTGGCTGGCTGACCCTG-3'.To determine the G894T polymorphism,two 17 bp ASO probes were used with the following sequences:5'-GATGAGCCCCCGAACT-3' and 5' GATGATCCCCCGAACT-3'.Detecting polymorphisms in the endothelial nitric oxide synthase gene scilicet genetype and allele frequency to undertake the study of case control.4.Statistics software SPSS 13.0 for statistical analysis:Continuous variables were compared with t-tests.Genotype and allele frequencies in controls and the two groups of cases were compared using standard x2 tests.Odds ratios and their confidence intervals were calculated according to Rothman or layering chi-square test.Results:1.The results of 3D-DSA,CT and TCCD detection:in 399 SAH patients,98 ASAH patients were suffered CVS,96 TSAH patients were suffered CVS,195 patients without CVS.2.We examined four eNOS polymorphisms,and two were associated with cerebral vasospasm in subarachnoid hemorrhage in the case-control comparisons:a T to C substitution in the promoter at position-786 and the a-deletion/b-insertion in intron. For the former,the risk of cerebral vasospasm was higher for C allele homozygotes than for the other two genotypes(odds ratio 2.68,95%CI,1.4 to 5.2).For the latter polymorphism,it was the a-deletion carriers that had the higher risk(odds ratio 2.3, 95%CI,1.3 to 3.8) in comparison with noncarriers.Both polymorphisms were analyzed together as haplotypes in a family-based study using the transmission disequilibrium test.The C/a-deletion haplotype was transmitted from heterozygous parents to cases with cerebral vasospasm in subarachnoid hemorrhage with a significantly higher frequency than expected(P=0.005).Conclusion:The findings of the case-control and family-based studies demonstrate clearly that DNA sequence differences in eNOS influence the risk of cerebral vasospasm in subarachnoid hemorrhage. Part 2 T-786C Polymorphism in Endothelial NO Synthase Gene Affects Cerebral Circulation in aneurysmal subarachnoid hemorrhage in SmokersObjective:To study the T-786C polymorphism in endothelial NO synthase gene affects cerebral circulation in aneurysmal subarachnoid hemorrhage in smokers.Method:Smokers and nonsmokers were defined as having a smoking index (cigarettes per day times years) of≥200 and 0,respectively.196 nonsmokers and 198 smokers in aneurysmal subarachnoid hemorrhage were recruited.1.Plasma was collected after overnight fasting to measure total cholesterol, HDL cholesterol,and triglyceride levels.2.Transcranial color duplex sonography(TCCD) detection:the imaging of the vascellum in the outside of cranium is formed by using the detecting head of pulse in 4～12MHZ,of intracalvarium in 2～4MHZ.The detecting head of TCCD being put on the segment inferior of cervical part,on the anterior border of stemomastoid muscle,on anodic of collarbone,or through temple window or pulvinar window detects the PSV,MVF and EDV in the ICA,ACA and MCA,respectively and records the PSV,MVF and EDV respectively.The patients of the three groups been detected once all in the third day and the seventh day differently of the posthemorrhagic.The formula Vm=Vs+(Vd×2)/3 for calculating MVF.3.The DNA of the peripheral blood of patients been extracted,by using the primers:C₀:TTTCTC CAG CCC CTCAGATG;2684C:GGC(?)GA GGC AGG GTC AG(?) CG2684T:CAT CAA GCT CTT CCC TG(?) CTT₀:AGG CCC AGC AAG GAT GTA GT,to amplify.Amplified by using PCR: Polymerase chain reaction(PCR) was performed in a total volume of 20μL containing 50 ng genomic DNA,0.25μmol/L 2684T and 2684C primers,0.063μmol/L To and Co primers,62.5μmol/L dNTPs,45 mmol/L Tris-HCl(pH 8.8),11 mmol/L ammonium sulfate,6.7μmol/Lβ-mercaptoethanol,4.5μmol/L EDTA,1.5 mmol/L MgCl₂,and 0.4 U Taq polymerase(Promega).After a hot start at 96℃, amplification was achieved by 35 cycles at 94℃for 30 seconds,60℃for 30 seconds, and 72℃for 20 seconds.Genotyping of T-786C was performed by using the PCR. Smokers were exposed to greater oxidative stress,as estimated by urinary F₂-isoprostane excretion.4.Statistics software SPSS 13.0 for statistical analysis.Results:1.The difference in body mass index was observed between the definite smokers and the nonsmokers(smoking group:BMI:23.51±3.17 kg/m²,nonsmoking group:BMI:24.36±3.37 kg/m²,P=0.011).Triglyceride levels were higher in the smokers(smoking group:TG:1.59±0.53 mmol/L,nonsmoking group:TG:1.37±0.49 mmol/L,P＜0.001 ).Urinary IsoP in the smokers was nearly double that in the nonsmokers(smoking group:1.75±0.63 ng/mg,nonsmoking group:1.14±0.32 ng/mg, P＜0.001).2.Two groups genotype frequencies did not differ significantly from one another(x²=0.894,df=2,P=0.640＞0.05).Analysising the influence of genetype of T-786C to mean arterial pressure(MAP),to MCA mean blood flow rate and to ICA mean blood flow indicate that In smokers,CC homozygotes of T-786C showed a significant increase of the mean flow velocity of arteria cerebri media(Vm:TT 95.8±56.3,TC 94.1±51.3,CC160.9±58.5 cm/s P＜0.001) and a significant increase of the mean flow velocity of arteria carotis interna(Vm:TT67.6±41.4,TC 64.9±42.2, CC120.7±67.7 cm/s P=0.018),where as in nonsmokers,the mean blood flow rates of MCA and ICA have no significant difference between the three genotypes,in MCA(Vm.TT 94.3±55.9,TC 93.6±57.9,CC 95.3±50.0 cm/s P=0.994),in ICA(Vm:TT 65.3±37.9,TC 64.7±33.7,CC 66.4±29.2 cm/s P=0.985).3.Mean arterial blood pressure of the smokers or nonsmokers has no significant difference,in smokers(TT 96.1±12.4,TC 94.9±12.2,CC 97.9±14.2,P=0.716),in nonsmokers(TT 97.2±12.8,TC 95.6±11.3,CC 96.7±12.2,P=0.754).Conclusion:The eNOS gene could modify cerebrovascular circulation in a general population by potentiating the adverse effect of smoking and speed up the cerebral blood flow rate.Part 3 The Research on the Dependability of Genetic Polymorphism of Haptoglobin and Aneurysmal Subarachnoid hemorrhageObjective:To investigate the distribution of serm haptoglobin phenotypes and gene frequency in patients of aneurysmal Subarachnoid hemorrhage(ASAH) and their relations with the severity of the disease.Method:Dividing into experiment group and control group,of the total,experiment group197 patients,98 patients with ASAH and CVS,99 patients with ASAH and without CVS,it is that the experiment group is divided into non-CVS group and CVS group,age 28～79,mean age(39.9±14.8),exclusiving hypertension,diabetes mellitus and cardiopathy;control group195 health adults,age 27～79,mean age(38±15.3), without hypertension,diabetes mellitus,angiocardiopathy and cerebrovascular diseases.Of the total,the CVS group is divided into three grades according to the degree of CVS:1 grade:partis vasospasm limits no more than 50%,or MCA≥120 cm/s,the ratio of MCA/ICA in 3～4,or DSA shows with CVS but the vestigial caliber exceed 50%;2 grade:partis vasospasm exceeds 50%,or MCA in 120～200 crn/s,the ratio of MCA/ICA in 4～6,or DSA shows with CVS but the vestigial caliber in 25%～50%;3 grade:diffuse and extentive CVS,or MCA≥200cm/s,the ratio of MCA/ICA≥6,or DSA shows with CVS and the vestigial caliber no more than 25%.The distribution of haptoglobin phenotypes in the serum of 197 SAH patients and 195 normal controls were determined by polyacrylamide gel vertical flat-bed electrophoresis.Results:(1) There was no significant difference between haptoglobin phenotypes and gene frequency among the patients with different severity of the disease(P＞0.05).(2) Compared with normal controls,Hp2-2 and Hp2-1 were significantly increased in non-CVS patient group(P＜0.05).Hp2 allele was also higher(P＜0. 05).(3) CVS patients showed increased frequency of Hp2-2,Hp2-1 and Hp2 allele, compared to non-CVS patients(P＜0.05).Conclusion:People with Hp2-1 and Hp2-2 may have genetic predisposition to SAH.Hp2 allele frequency may assemble in non-CVS and CVS patients and relate to the increased genetic predisposition of the disease,which may play a role in the pathogenesis of SAH and CVS.Part 4 Association Between Polymorphism of Apolipoprotein E gene And Aneurysmal Subarachnoid hemorrhageObjective:To study the association between polymorphism of apolipoprotein E gene,aneurysmal subarachnoid hemorrhage and cerebral vasospasm.Method:1.Test groups:divide into experiment group and control group,of the total, experiment group197 patients,98 patients with ASAH and CVS,99 patients with ASAH and without CVS,it is that the experiment group is divided into SAH group and CVS group,age 28～79,mean age(39.9±14.8),exclusiving hypertension,diabetes mellitus and cardiopathy;control group195 health adults,age 27～79,mean age (38±15.3),without hypertension,diabetes mellitus,angiocardiopathy and cerebrovascular diseases.All the patients are carried out CT scanning in admission and according to Fisher's grading system,Hunt and Hess grading scale and World Federation of Neurosurgical Societies(WFNS) grading scale,undertake grouping; Outcome was assessed with the Glasgow Outcome Scale after 6 monthes of postoperative.2.Specimen collection:all the patients fasting diet 12 h,drawed 5ml the blood of empty stomach,of the blood,2ml(EDTA anticoagulation) was used to extract DNA routinely,to undertake analysis of polymorphism of APOE gene;3ml blood was used to detect the biochemical indicator of blood fat,blood sugar etc.3.Blood fat and apoprotein detect:after collecting blood preparation,to detect TC,TG,LDL-C,HDL-C,ApoA and ApoB in same automatic biochemistry analyzer.4.Genome DNA extraction:extracting specimen peripheral blood genome DNA for PCR amplifying,primer upstream sequence is 5'-TCCAAGGAGCTGCAGGCGGCGCA-3',primer downstream sequence is 5'-ACAGAATTCGCCCCGGCCTGGTACACTGCCA--3'.5.PCR amplifying and Cfol incision enzyme digestion:Polymerase chain reaction(PCR) was carried out in a 20μL volume with 200 ng of genomic DNA,10 pmol of each primer,100μmol/L dNTP,1 units of display TAQ DNA Polymerase with 5μL of 10×PCR buffer containing 2.5 mmol/L MgCl₂,10%DMSO(V/V).Initial denaturation was performed at 95℃for 35 seconds and was followed by 30 cycles consisting of 60℃for 35 seconds,70℃for one minute,and amplification was finished with extension at 72℃for ten minutes.Restricted enzyme incide reaction was undertaken in a 20μL volume containing PCR reaction product 12μL,Cfol incision enzyme 8U(10U/μl),mix even lightly and stay overnight in 37℃,digested products to be undergone 8%polyacrylamide gel electrophoresis.6.Silver staining:after electrophoresis,gel was washed 2～3 times by distilled water,silver staining 8 min using 0.2%AgNO3.Flushing gel several times using distilled water,coloration succi deoxidize silver granules until strap clear.Classify APOE genotype according to electrophoregram.7.Handling with statistics:detection results were dealed with SPSS13.0 statistics software,ratio was compared using standard x² tests,interclass mean were compared with t-tests.Results:1.Primary clinical condition and radiological findings were quite similar between the APOE e4-negative and APOEe4-positive groups.2.A total of 23(34%) of the 68 patients with APOE e4 had an unfavorable outcome compared with 27(21%) of the 129patients without APOE e4,which between the two groups of comparison had significant diference(Z=-1.972,P=0.049). The patients with e4 allele had an unfavorable outcome easily.3.Of CVS group APOE4/3 genotype and frequency of E4 allele was significant high than control group(P＜0.01 and P＜0.05).4.The level of TC,LDL-C and APOB of Experiment group were significant high than of control group,where as the level of HDL-C and APOA1 were significant low than of control group.5.Among the subsets of different APOE genotypes,the level of TC,LDL-C and APOB was decrement from E4 subset to E3 subset to E2 subset;the level of HDL-C and APOA1 was increasing progressively from E4 subset to E3 subset to E2 subset. E4 allele carriers had high level of TC,LDL-C and APOB;where as E2-allele-carriers had high level of HDL-C and APOA1 than E3-allele-carriers and E4-allele-carriers.Conclusion:There is a significant association between APOE polymorphism and cerebral vasospasm after aneurismal SAH.