Evaluation Of Carotid Plaque Vulnerability By Contrast-enhanced Ultrasound Imaging:a Clinical Study Based On Histopathology

This study consists of three parts:(1)Evaluation of carotid plaque vulnerability by contrast-enhanced ultrasound imaging and histopathology;(2)The association between carotid plaque vulnerability and ischemic stroke;(3)The association between cardiovascular risk factors and carotid plaque vulnerability.Part Ⅰ Evaluation of carotid plaque vulnerability by contrastenhanced ultrasound imaging and histopathologyObjective This study aims to assess the value of CEUS for the detection of plaque cap rupture and neovascularization in histologically verified plaques that had been removed from the patients who had undergone carotid endarterectomy(CEA).Methods1.Data:Consecutive patients underwent CEA in the Stroke Center of the first affiliated Hospital of Soochow University from January 2019 to December 2019 were recruited in this study.The inclusion criteria were symptomatic patients with carotid stenosis more than 50%or asymptomatic patients with more than 70%lumen stenosis.The exclusion criteria were patients with fragmented plaques caused by the operation,plaques with severe calcification,patients with the acute coronary syndrome,patients with acute heart failure,patients with unstable angina,patients who were allergic to contrast media,patients with severe intraoperative middle cerebral artery stenosis and patients with posterior circulation infarction.In this study,51 CEA patients(51 plaques)met the inclusion and exclusion criteria.2.Contrast-enhanced ultrasound:(1)patients underwent contrast-enhanced ultrasound before operation.The direction of the contrast agent diffusion within plaques was classified as "inside-out"direction(contrast medium perfusion from carotid lumen to plaque)and "outside-in" perfusion mode(contrast medium perfusion from carotid adventitia to plaque).Inside indicates lumen,outside indicates adventitia.The "inside-out" perfusion mode includes two manifestations:the typical ulcer type,the contrast medium flows into the plaque ulcer from the lumen,that is,the contrast medium fills the plaque ulceration and the fissure type,the microbubbles spread from the carotid lumen to the plaque in a moving line.(2)the enhancement degree of contrast-enhanced ultrasound in the plaque can be divided into grade 1:no enhancement in the plaque,that is,no bubbles in the plaque,or adventitia enhancement;grade 2:moderate enhancement in the plaque,a small number of moving microbubbles confined in the shoulder of the plaque.Grade 3:extensive enhancement in the plaque,and moving microbubbles were widely concentrated in the core of the plaque.3.Pathological examination:the characteristics of plaque vulnerability were evaluated according to the pathological standard after the operation,including fibrous cap rupture,intraplaque hemorrhage,thrombosis,large lipid core,and plaque microvessels.The correlation between contrast-enhanced ultrasound perfusion mode and plaque vulnerability was analyzed.4.Statistical methods:independent sample t-test was used to analyze the continuous variables with normal distribution between the two groups,Mann-Whitney U test was used to analyze the continuous variables with non-normal distribution between the two groups,and x2 test or Fisher accurate test was used to analyze the classified variables between the two groups.The sensitivity,specificity,positive predictive value,and negative predictive value of contrast-enhanced ultrasound in the diagnosis of plaque fibrous cap rupture were evaluated.After adjusting the confounding factors of age,sex,and carotid stenosis,Logistic regression analysis was used to evaluate the correlation between the "inside-out" perfusion pattern of contrast-enhanced ultrasound and plaque fibrous cap rupture.Results1.51 patients with CEA(51 plaques)were included in this study,and 23 plaques(45.1%)performed "inside-out" direction perfusion mode.28 plaques(54.9%)performed"outside-in" direction perfusion mode.Histopathological examination showed that 24(47.1%)plaques with fibrous cap rupture,29(56.9%)cases with intraplaque hemorrhage,13(25.5%)cases with thrombosis,and 28(54.9%)with large lipid core.2.The correlation between contrast-enhanced ultrasound perfusion mode and fibrous cap rupture:the incidence of fibrous cap rupture in plaques with "inside-out" perfusion mode was significantly increased(73.9%vs 25.0%,p<0.001).The sensitivity,specificity,positive predictive value,and negative predictive value of contrast-enhanced ultrasound "inside-out"perfusion mode in detecting plaque fibrous cap rupture were 87.5%,92.6%,91.3%,and 89.3%,respectively.After adjusting for confounding factors of age,gender,and carotid stenosis,logistic regression analysis showed that the contrast-enhanced ultrasound "insideout" perfusion mode was an independent risk factor for plaque fibrous cap rupture(OR 8.5,95%Cl 2.4-30.1,p=0.001).3.The correlation between the enhancement degree of and neovascularization:in fibrous cap ruptured plaques,there was no significant correlation between the enhancement degree of and microvessels density(p=0.131),but intact plaques,the enhancement degree was positively correlated with the microvessel density(p=0.001).Conclusion The "inside-out" perfusion mode can indicate plaque fibrous cap rupture;and in plaques with fibrous cap rupture,contrast enhancement can not accurately assess neovascularization.Part Ⅱ The association between carotid plaque vulnerability and ischemic strokeObjective This study aims to evaluate the correlation between ischemic stroke and carotid plaque vulnerability to explore the "high risk" risk markers associated with ischemic stroke.Methods1.The inclusion and exclusion criteria,examination methods and image analysis were the same as the first part.Grouping criteria and pathological basis:according to whether the patients had ischemic stroke within six months,patients were divided into symptomatic group and asymptomatic group.2.Histological examination:After CEA,the vulnerable characteristics of plaques were evaluated according to the pathological gold standard,such as fibrous cap rupture,intraplaque hemorrhage,thrombosis,large lipid core and microvessels,and the correlation between plaque vulnerability and ischemic stroke was analyzed.3.Statistical methods:independent t-test was used to analyze the continuous variables with normal distribution between the two groups,Mann-Whitney U test was used to analyze the continuous variables with non-normal distribution between the two groups,and χ2 test or Fisher accurate test was used to analyze the classified variables between the two groups.Results1.Distribution of patients in symptom group and asymptomatic group:51 CEA patients met the inclusion and exclusion criteria.42(82.4%)patients were divided as ischemic stroke group,showing weakness or even hemiplegia of one limb,transient ischemic attack,transient amausia and inarticulate speech.9(17.6%)patients in the asymptomatic group were treated with CEA because of severe carotid stenosis.Histopathological examination showed fibrous cap rupture in 47.1%(24/51)plaques,intraplaque hemorrhage in 56.9%(29/51)plaques,large lipid core in 54.9%(28/51)plaques,and thrombus in 25.5%(13/51)plaques.2.Symptom group and asymptomatic group:The incidence of carotid plaque fibrous cap rupture was significantly higher in the symptomatic group than in the asymptomatic group(54.8%vs 11.1%,p=0.017).Carotid plaques in patients with ischemic stroke were more likely to have thrombus(31.0%vs 0%,p=0.053)and large lipid core(61.9%vs 22.2%,p=0.061),but the difference was not statistically significant.There was no significant difference between symptomatic and asymptomatic patients in intraplaque hemorrhage(61.9%vs 33.3%,p=0.116)and plaque microvessel density(3.2±2.0 vs 2.6±3.5,p=0.544).3.The correlation between plaque vulnerability assessed by contrast-enhanced ultrasound and ischemic stroke:the incidence of "inside-out" perfusion mode in symptomatic patients was significantly higher than that of asymptomatic patients(52.4%vs 11.1%,p=0.024).However,there was no significant difference in the degree of contrastenhanced plaque between symptomatic and asymptomatic patients(p=0.649).After excluding carotid atherosclerotic plaques with ruptured fibrous cap,in 27 plaques with intact fibrous cap,there was no significant difference in enhancement degree of carotid plaques between symptomatic and asymptomatic patients(p=0.535).Conclusion The "inside-out" perfusion mode of contrast-enhanced ultrasound is a "high risk"imaging marker closely related to ischemic stroke.The enhancement degree of carotid plaqeu does not fully indicate the risk of ischemic stroke.Part Ⅲ The association between cardiovascular risk factors and carotid plaque vulnerabilityObjective Cardiovascular risk factors such as hypertension can cause carotid atherosclerotic plaque formation and carotid artery stenosis,but whether it will affect plaque vulnerability is rarely reported.This study aims to evaluate the association between cardiovascular risk factors and carotid plaque vulnerability to provide evidence for prevention and treatment of stroke in patients with atherosclerotic disease.Methods 1.Consecutive patients underwent carotid endarterectomy(CEA)from January 2019 to July 2021 in the stroke center of the first affiliated hospital of Soochow University were recruited.The inclusion criteria were the same as those in the first part.The exclusion criteria were patients with fragmented plaques caused by the operation,plaques with severe calcification,patients with the acute coronary syndrome,patients with acute heart failure,patients with severe intraoperative middle cerebral artery stenosis and patients with posterior circulation infarction.128 CEA patients(128 plaques)met the inclusion and excusion criteria.The clinical data and biochemical examination of the patients were collected within 24 hours after admission.Cardiovascular risk factors include hypertension,diabetes,dyslipidemia,smoking,hyperhomocysteinemia and history of coronary heart disease.2.Pathological analysis:firstly,the characteristics of plaque vulnerability were evaluated according to the pathological gold standard after CEA,including fibrous cap rupture,intraplaque hemorrhage,thrombosis and large lipid core in the plaque.Secondly,the overall vulnerability of plaques is evaluated according to the semi-quantitative classification criteria of Oxford Plaque Study.Probably unstable plaques and absolutely unstable plaques were classified as vulnerable plaques.3.Statistical methods:Logistic regression analysis was used to evaluate the risk factors independently associated with overall plaque vulnerability,and evaluate the risk factors independently associated with plaque vulnerable characteristics(thrombosis,plaque rupture,intraplaque hemorrhage and large lipid core).The correlation between hypertension and plaque fibrous cap rupture was analyzed by Logistic regression analysis.The patients were divided into three groups at the tertile of systolic pressure,diastolic blood pressure and pulse pressure.Firstly,the systolic blood pressure was divided into three groups:<133mmHg,133-146mmHg,>146mmHg,<133mmHg level group as the reference group.Secondly,the diastolic blood pressure was divided into three groups:<73 mmHg,73-80 mmHg,>80 mmHg,<73 mmHg as the reference group.Finally,pulse pressure was divided into three groups:<57 mmHg,57-68 mmHg,>68 mmHg,<57 mmHg as the reference group.The regression model includes uncorrected model and multivariate corrected model,age and sex were adjusted.Results 1.A total of 128patients with CEA were included in this study,including 73 patients with hypertension(57.0%),79 patients with dyslipidemia(61.7%)and 75 patients with diabetes(58.6%).There were 48 smokers(37.5%).Histopathological examination showed fibrous cap rupture in 35.9%(46/128)plaques,intraplaque hemorrhage in 54.7%(70/128)plaques,large lipid core in 47.7%(61/128)plaques,and thrombus in 21.9%(28/128)plaques.2.The correlation between cardiovascular risk factors and overall plaque vulnerability:hypertension(OR 5.97,95%Cl 1.96-18.20,p=0.002)and dyslipidemia(OR 3.82,95%Cl 1.32-11.09,p=0.014)were closely related to plaque overall vulnerability.3.The correlation between cardiovascular risk factors and plaque vulnerable characteristics:Hypertension was an independent risk factor for plaque fibrous cap rupture(OR 6.12,95%CI 2.32-16.17,p<0.001)and intraplaque hemorrhage(OR 3.54,95%CI 1.55-8.06,p=0.003).Patients with hypertension(OR 2.34,95%CI 1.05 5.18,p=0.037)and with dyslipidemia(OR 3.22,95%CI 1.41-7.34,p=0.006)had a significantly increased risk of developing a large lipid core.Smoking is closely related to thrombosis(OR 6.23,95%CI 2.31-16.80,p<0.001).4.The correlation between hypertension and plaque fibrous cap rupture:After adjusting for the confounding factors of age and sex,the risk of fibrous cap rupture in patients with systolic blood pressure 133-146 mmHg group(OR 3.39,95%CI 1.18-9.73,p=0.023)and>146 mmHg group(OR 5.61,95%Cl 2.01-15.64,p=0.001)was significantly increased.After adjusting for the confounding factors of age and sex,the risk of fibrous cap rupture in patients with pulse pressure 57-68 mmHg group(OR 7.82,95%CI 2.48-24.69,p<0.001)and>69mmHg group(OR 9.58,95%Cl 3.06-30.01,p<0.001)was significantly increased.When diastolic blood pressure was used as classified variable,there was no significant difference between diastolic blood pressure and plaque fibrous cap rupture(p>0.05).Conclusion Cardiovascular risk factors can affect the vulnerability of carotid plaque,hypertension is the most significant risk factor for plaque fibrous cap rupture.