| BackgroundStroke,of which ischemic stroke（IS）accounts for about 84.4%,is the leading cause of death and lifetime disability in China as well as one of the major causes of mortality and disability worldwide.Reperfusion of the ischemic brain tissue,through prompt recanalization of occluded cerebral vessels,is the most effective therapy of acute ischemic stroke（AIS）.Intravenous（Ⅳ）thrombolysis with recombinant tissue plasminogen activator（rt-PA）within 4.5 hours after onset has been shown to improve neurological sequelae in appropriate AIS patients in a number of clinical studies.Hemorrhagic transformation（HT）is the main potential complication and a major limitation of rt-PA treatment in AIS.Symptomatic intracranial hemorrhage（sICH）,85-90%of which occurs within 24 hours after Ⅳ rt-PA,causes neurological deterioration with an incidence of 2-7%.Mechanisms underlying HT after Ⅳ rt-PA are complicated and include reperfusion injury,blood-brain barrier（BBB）disruption,and rt-PA-related coagulopathy,among others.Homocysteine（Hcy）,a sulfur-containing amino acid,is mainly produced by methionine metabolism.Its blood concentration is influenced by multiple factors,including age,sex,lifestyle factors,B vitamin deficiencies,genetic defects impairing the activity of enzymes involved in Hcy metabolism,certain drugs and diseases.The normal level of blood Hcy is 5-15 μmol/L and an abnormally elevated level≥15μmol/L is defined as hyperhomocysteinemia（HHcy）,which affects approximately 27.5%of the general population in China.Initially proposed to induce arteriosclerosis by McCully et al in 1969,Hcy has now been reported to be associated with various diseases of the central nervous system（CNS）,including diverse cerebrovascular diseases,Parkinson’s disease,amyotrophic lateral sclerosis,Alzheimer’s disease and migraine.As a potentially modifiable risk factor for IS,Hcy may also play a role in intracranial hemorrhage,intracranial aneurysm and cerebral small vessel disease（including cerebral microbleeds）.Besides,Hcy has been revealed to contribute to unfavorable outcome of AIS patients in some studies.However,little is known about the effects of serum Hcy level on HT and clinical outcome in AIS patients subjected to Ⅳ rt-PA.Therefore,part Ⅰ and part Ⅱ of the study aimed to investigate whether serum Hcy level was related to HT within 24 hours of Ⅳ rt-PA and clinical outcome at 3 months through respectively analyzing data of AIS patients submitted to Ⅳ rt-PA within 4.5 hours of symptom onset.Although BBB disruption is one of the main mechanisms of HT and unfavorable prognosis after rt-PA treatment in AIS patients,there has been no research investigating the impacts of mild HHcy on BBB integrity after Ⅳ rt-PA in AIS patients so far.Therefore,immortalized murine brain-derived endothelial cells（bEnd.3）was used as the in vitro model of BBB and submitted to glucose oxygen deprivation/re-oxygenation（OGD/R）in combination with rt-PA to explore the underlying mechanisms of serum Hcy increasing the risk of HT and unfavorable outcome after Ⅳ rt-PA in AIS patients in part Ⅲ of the study.Part ⅠStudy on the association between serum Hey and HT after Ⅳ rt-PA in AISpatientsObjectiveThere have been only a few studies concerning the association between serum Hcy level and HT in AIS patients submitted to Ⅳ rt-PA with controversial results.The first part of the study aims to investigate whether serum Hcy level contributes to HT susceptibility within 24 hours of Ⅳ rt-PA in AIS patients.MethodsThe following data of 236 consecutive AIS patients submitted to rt-PA treatment within 4.5 hours of symptom onset were collected and analyzed retrospectively:age,sex,medical history related to AIS,smoking,alcoholism,medication history,door-to-needle time（DNT）,onset-to-needle time（ONT）,blood pressure and the National Institutes of Health Stroke Scale（NIHSS）score before rt-PA administration,NIHSS score 24 hours after rt-PA administration,laboratory measures（including serum Hcy levels）,cranial computed tomography（CT）and/or magnetic resonance imaging（MRI）and examinations of intracranial and extracranial vessels,examination of the heart（routine electrocardiogram,echocardiogram and ambylatory electrocardiogram,etc.）as well as the Trial of Org 10172 in Acute Stroke Treatment（TOAST）classification.Cranial imaging was evaluated retrospectively for HT within 24 hours of Ⅳ rt-PA and apparent hemorrhage inducing clinical deterioration with a≥4-point increase on NIHSS score or death was diagnosed as sICH.All subjects were divided into two groups based on whether they suffered from HT and sICH within 24 hours of Ⅳ rt-PA,respectively.The aforementioned data were compared between the HT group and the non-HT group as well as between the sICH group and the non-sICH group.Spearman correlation analysis was applied to explore the correlation between serum Hcy level and NIHSS score before rt-PA treatment,A multivariable logistic regression analysis was used to assess whether there was an independent effect of serum Hcy level on HT within 24 hours of Ⅳ rt-PA in AIS patients and the results were presented as odds ratio（OR）and 95%confidence interval（CI）.The receiver operating characteristic curve（ROC）and the area under the curve（AUC）were employed to evaluate the performance of serum Hcy level in predicting HT within 24 hours of Ⅳ rt-PA in AIS patients and and the optimal cut-off point was calculated by maximizing Youden’s index.IBM SPSS software version 25.0 was applied and significance was set at P<0.05.Results1.There were 169 men（71.6%）out of all 236 subjects,aged 32.0 to 88.0（median 65.0）years.Massive cerebral infarctions were identified in 23 patients（9.7%）.Twenty-eight patients（11.9%）presented with HT within 24 hours of rt-PA treatment,and sICH occurred in 7 patients（3.0%）.Serum Hcy levels ranged from 4.45 to 67.71（median 12.05）μmol/L and pre-treatment NIHSS scores ranged from 0 to 28.0（median 5.0）.The proportions of each subtype in TOAST classification were as follows:large-artery atherosclerosis（LAA）49.2%,cardioembolism（CE）10.6%,small artery occlusion（SAO）32.2%,stroke of other determined etiology（SOE）1.3%and stroke of undetermined etiology（SUE）6.8%.Serum Hcy levels were not correlated with pre-treatment NIHSS scores.2.Compared with non-HT patients,serum Hcy levels（13.00 vs.11.70 μmol/L）,pre-treatment NIHSS scores（9.0 vs.5.0）and proportion of massive cerebral infarction（21.4%vs.8.2%）were higher in patients with HT.While,proportion of SAO in TOAST classification was lower in patients with HT than in those without HT（7.1%vs.35.6%）.The multivariable logistic regression analysis indicated that serum Hcy level as a continuous variable was independently related to an increased risk of HT within 24 hours of Ⅳ rt-PA in AIS patients with an adjusted OR of 1.103（95%CI=1.021-1.191,P=0.013）.3.Serum Hcy（15.19 vs.11.73 μmol/L）and prothrombin time（13.3 vs.12.1 sec）levels,pretreatment NIHSS scores（15.0 vs.5.0）,proportions of CE in TOAST classification（42.9%vs.9.6%）as well as massive cerebral infarction（57.1%vs.8.3%）were remarkably higher in sICH patients than in non-sICH ones.4.HT risk within 24 hours of Ⅳ rt-PA increased as the serum Hcy level increased,as illustrated in the ROC curve with an AUC value of 0.630（95%CI=0.530-0.730,P=0.025）.The optimal cut-off value of 11.88μmol/L had a sensitivity of 71.4%and a specificity of 51.9%.Conclusions1.The incidence rates of HT and sICH are 11.9%and 3.0%within 24 hours ofⅣ rt-PA in AIS patients,respectively.2.Serum Hcy levels are significantly higher in AIS patients suffering from HT within 24 hours of Ⅳ rt-PA than in those without HT.Similarly,patients presenting with sICH within 24 hours of Ⅳ rt-PA have higher serum Hcy levels than those without sICH.3.Serum Hcy level is an independent predictor for HT within 24 hours of Ⅳrt-PA in AIS patients.Part ⅡStudy on the association between serum Hcy and clinical outcome after Ⅳ rt-PA in AIS patientsObjectiveStudies about the association between blood Hcy concentrations and prognosis of AIS patients have not reached a consistent conclusion.Ⅳ rt-PA has been verified to improve clinical outcome at 3 months in appropriate AIS patients.The second part of the study aims to investigate whether serum Hcy level contributes to unfavorable outcome at 3 months in AIS patients submitted to rt-PA treatment.MethodsThe following data of 184 consecutive AIS patients submitted to Ⅳ rt-PA within 4.5 hours of symptom onset and followed up for 3 months were colletcted and analyzed retrospectively:age,sex,medical history related to AIS,smoking,alcoholism,NIHSS scores before and 24 hours after rt-PA administration,DNT,ONT,laboratory measures（including serum Hcy levels）,TOAST classification and the modified Rankin Scale（mRS）score obtained at 90±7 days.All subjects were categorized into two groups on the base of mRS scores at 3 months:the favorable outcome group（mRS score 0-2）and the unfavorable outcome group（mRS score 3-6）.The aforementioned data were compared between these two groups.Spearman correlation analysis was used to explore the correlation between serum Hcy level and NIHSS score before rt-PA treatment.A multivariable logistic regression analysis was applied to assess whether there was an independent effect of serum Hcy level on unfavorable outcome at 3 months of AIS patients submitted to Ⅳrt-PA.The ROC and AUC were employed to evaluate the performance of serum Hcy level in predicting unfavorable outcome at 3 months of AIS patients submitted to rt-PA treatment and the optimal cut-off point was calculated by maximizing Youden’s index.IBM SPSS software version 25.0 was applied and significance was set at P<0.05.Results1.There were 140 men（76.1%）out of all 184 subjects,aged 32.0-87.0（median 65.0）years.128 subjects（69.6%）presented with favorable outcome and 56 subjects（30.4%）presented with unfavorable outcome（12 patients died）at 3 months.NIHSS scores before rt-PA administration and 24 hours after rt-PA treatment ranged from 0-28.0（median 5.0）and 0-25.0（median 3.0）,respectively.Serum Hcy levels ranged from 4.45 to 67.71（median 12.30）μmol/L.The proportions of each subtype in TOAST classification were as follows:LAA 47.8%,CE 12.5%,SAO 32.1%,SOE 1.6%and SUE 6.0%.Serum Hcy levels were not correlated with pre-treatment NIHSS scores.2.Compared to patients with favorable outcome,patients with unfavorable outcome had higher serum Hcy levels（12.74 vs.11.98 μmol/L）as well as higher NISSS scores before rt-PA administration（12.0 vs.4.0）and 24 hours after rt-PA treatment（11.0 vs.2.0）.Moreover,proportions of subjects complicated with hypertension（71.4%vs.54.7%）,coronary artery disease（30.4%vs.10.9%）and atrial fibrillation（28.6%vs.9.4%）in the unfavorable outcome group far exceeded those in the favorable outcome group.Etiological subtype distribution（TOAST classification）also differed between the two subgroups,with a notably higher proportion of CE（26.8%vs.6.3%）and a markedly lower proportion of SAO（17.9%vs.38.3%）in patients with poor outcome.3.Whether NIHSS scores before Ⅳ rt-PA（OR=1.109,95%CI=1.018-1.207,P=0.017）or NIHSS scores 24 hours after Ⅳ rt-PA（OR=1.212,95%CI=1.039-1.413,P=0.014）were included in the multivariable logistic regression analysis,serum Hcy level as a continuous variable was independently associated with an increased risk of unfavorable outcome at 3 months in AIS patients submitted to rt-PA treatment.4.The risk of poor outcome at 3 months of AIS patients submitted to Ⅳ rt-PA increased as the serum Hcy level increased,as illustrated in the ROC curve with an AUC value of 0.601（95%CI=0.514-0.688,P=0.029）.The optimal cut-off value of 12.05 μmol/L had a sensitivity of 64.3%and a specificity of 50.8%.Conclusions1.The incidence rate of unfavorable outcome at 3 months in AIS patients submitted to rt-PA treatment is 30.4%.2.Serum Hcy levels in rt-PA-treated AIS patients suffering from unfavorable outcome at 3 months are significantly higher than in those with favorable outcome.3.Serum Hcy level is an independent predictor for unfavorable outcome at 3 months in AIS patients receiving rt-PA treatment.Part ⅢStudy on the related mechanisms of serum Hcy increasing the risk of HT and unfavorable outcome after Ⅳ rt-PA in AIS patientsObjectiveBBB leakage is one of the main mechanisms underlying HT and poor outcome in AIS patients submitted to rt-PA treatment.The third part of the study aims to explore the underlying mechanisms of serum Hcy increasing the risk of HT and unfavorable outcome after Ⅳ rt-PA in AIS patients.MethodsBEnd.3 cells were used as the in vitro model of BBB and OGD/R was performed on bEnd.3 cells to establish the in vitro model of ischemia-reperfusion.Based on different interventions（30 μmol/L Hcy,OGD/R,20 μg/mL rt-PA）,bEnd.3 cells were randomly assigned to one of the following five groups in the first stage:control,OGD/R,Hcy+OGD/R,OGD/R+rt-PA and Hcy+OGD/R+rt-PA.Pare-cellular permeability of cells were determined 24 hours after re-oxygenation through measurement of the diffusion of fluorescein sodium salt（NaF）.Moreover,cells were harvested 24 hours after re-oxygenation for protein and mRNA extraction.Protein levels of occludin,claudin-5,matrix metalloproteinase-9（MMP-9）,extracellular regulated protein kinases（ERK）1/2 as well as phosphorylated-ERK1/2（p-ERK1/2）（Thr202/Tyr204）were detected through western blot analysis.MMP-9 mRNA levels were assessed through quantitative reverse transcription-polymerase chain reaction（qRT-PCR）.In the second stage,bEnd.3 cells were randomly divided into to four groups:OGD/R+rt-PA,Hcy+OGD/R+rt-PA,Hcy+DMSO+OGD/R+rt-PA and Hcy+U0126+OGD/R+rt-PA,based on different interventions（30 μmol/L Hcy,DMSO,10 μmol/L U0126,OGD/R,20 μg/mL rt-PA）.The detection methods were the same as those in the first stage.Comparison of different groups was performed by one-way analysis of variance（ANOVA）followed by Tukey post-hoc corrections using Graphpad Prism 8.0 and significance was set at P<0.05.Results1.Exposure to OGD for 4 hours and re-oxygenation for 24 hours resulted in a notable increase in passage of NaF across the bEnd.3 monolayers by 49.6%and marked decreases in protein levels of occludin and claudin-5.Pre-treatment with 30μmol/L Hcy for 24 hours potently exacerbated para-cellular leakage（171.2%）and induced a further decrease of occludin protein level instead of claudin-5 protein level in bEnd.3 cells submitted to OGD/R.The presence of rt-PA at re-oxygenation further increased the passage of NaF（191.8%）and decreased protein levels of occludin and claudin-5.30μmol/L Hcy aggravated para-cellular leakage（254.7%）and tight junction disruption in OGD/R+rt-PA-injured bEnd.3 cells.2.OGD for 4 hours remarkably increased phosphorylation of ERK1/2 as well as MMP-9 protein and mRNA levels in bEnd.3 cells.Both 30 μmol/L Hcy and rt-PA induced further increases in phosphorylation of ERK1/2 and MMP-9 expression levels in OGD/R-treated bEnd.3 cells.Administration of 30 μmol/L Hcy for 24 hours before OGD strikingly activated the ERK signal transduction pathway and upregulated MMP-9 expression levels in OGD/R+rt-PA-injured bEnd.3 cells.3.U0126 administration before OGD efficaciously deactivated the ERK signal transduction pathway,repressed MMP-9 expression and alleviated the deleterious effects of 30 μmol/L Hcy on tight junctions and para-cellular permeability in OGD/R+rt-PA-injured bEnd.3 cells.Conclusions1.In the in vitro ischemia-reperfusion model,30 μmol/L Hcy further increased para-cellular pearmeability of rt-PA-treated bEnd.3 cells.2.In the in vitro ischemia-reperfusion model,30 μmol/L Hcy activated the ERK signal transduction pathway in rt-PA-treated bEnd.3 cells,and then up-regulated the expression level of MMP-9 and aggravated the disruption of tight junctions. |
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