Research On The Vulnerability Of Carotid Atherosclerotic Plaque By Histopathological Analysis Based On Ultrasound Features Multiparameter

Part Ⅰ The assessment of carotid atherosclerosis plaque vulnerability by ultrasound features multiparameter and histopathologyObjectiveTo explore the ability of ultrasound features multiparameter and histolpathology in evaluating carotid artery atherosclerosis plaque vulnerability,and the difference in different parts of the plaque.Methods1.Reseach subjects:This research continuously included 55 patients(55 plaques)undergoing carotid endarterectomy(CEA)in stroke centers from January 2017 to August 2018 at the First Affiliated Hospital of Soochow University,50 males and 5 females,mean age 64.1±7.3 years.CEA surgery is indicated following the North American Standard for Symptomatic Carotid Endarterectomy(NASCET):patients with carotid luminal stenosis≥70%without symptoms of ischemic stroke,or patients with luminal stenosis rate≥50%and with symptoms of ischemic stroke.Inclusion criteria:(1)patient with moderate or above vascular stenosis of internal carotid artery who met NASCET criteria for proposed CEA surgery;(2)Carotid ultrasound before CEA surgery and retention of complete images;(3)CEA surgery within 24 hours after ultrasound exam.Exclusion criteria:(1)Incomplete carotid atherosclerotic culprit plaque specimen obtained after surgery;(2)Plaque’s HE staining failure;(3)Lack of standard carotid ultrasound images;(4)Lack of complete history or serum test information.2.Carotid artery ultrasound exam:carotid artery ultrasound exam were performed on the patients before carotid endarterectomy.The ultrasound features multiparameter(carotid artery atherosclerosis plaque length,thickness,shape,echo,ulcer,stenosis rate and resistance index of stenosis)were observed and recorded.The ultrasound features scores were calculated:plaque shape(0 points for shape rules and 1 point for irregular shape);internal echo of plaques(0 points for high echo gauge for plaque,1 point for mixed echo gauge for plaque,and 2 points for low echo for plaque);Whether there is an ulcer in the plaque(0 points for no ulcer in the plaque,1 point for the presence of ulcers);Vascular stenosis rate(vascular stenosis rate<50%is mild stenosis and counts as 0 points,vascular stenosis rate of 50%to 69%is moderate stenosis and counts as 1 point,vascular stenosis rate of 70%-89%is severe stenosis is 2 points,vascular stenosis rate ≥ 90%is very severe stenosis and counts 3 points).The ultrasound feature parameters of the upstream(proximal heart segment)and the downstream side(distal segment)of the plaque were observed,and the ultrasound feature scores of different sites were counted.3.Pathological examination:Each plaque was continuously cross-sectioned sectionally sectioned with a thickness of 4 μm at 5 mm around the narrowest part of the carotid artery,stained with hematoxylin-eosin(HE).Different parts of all plaques(upstream and downstream sides 3-5 mm from the plaque migration)are also made in continuous crosssectional sections of 4 μm thickness and HE stained.Histopathological features were observed under the microscopy,and all the carotid artery atherosclerosis plaques were grouped to vulnerable plaque group or stable plaque group by according to Naghavi M.and others.4.Statistical methods:Comparing the measurement data of plaque length,plaque thickness,vascular stenosis rate,and resistance index at stenosis between vulnerable plaque and stable plaque groups,one-way ANOVA was used.Comparison of ultrasound feature scores between the two groups was performed using the Mann-Whitney U test.Subjects’Working Characteristic Curve(ROC)assesses the performance of ultrasound signature scoring in diagnosing vulnerable plaques.The ultrasonic feature scores on the upstream and downstream sides of the plaque were compared using the Wilcoxon symbolic rank and test.The chi-square test was used for counting data comparison.Results1.Grouping:Among the 55 cases,76.4%(42/55 cases)were in the vulnerable plaque group and 23.6%(13/55 cases)were in the stable plaque group.2.Comparison of ultrasound feature parameters between two groups:The ultrasound features scores of vulnerable plaque group were significantly higher than those of stable plaque group(P=0.001).The plaques of vulnerable plaque group were significantly thicker than those of stable plaque group(P=0.001).The stenosis rates of vulnerable plaque group were significantly higher than those of stable plaque group(P=0.047).On the other hand,there was not statistically significant diffence of the plaque lengthes and resistance indexes between vulnerable plaque group and stable plaque group(P=0.063 and P=0.459).ROC curve analysis:the AUC of ultrasound features score’s ability to distinguish vulnerable plaque and stable plaque was 0.878 and cut-off point was 3;the AUC of plaque thickness was 0.853 and cut-off point was 4.65mm;the AUC of stenosis rate was 0.739 and cut-off point was 72.5%.3.Ultrasound characteristic parameters of different parts of the plaque:There were significantly more irregularity in upstream sides than in downstream sides(P=0.001).There were more lesions or ulcers in upstream sides than in downstream sides(P=0.003).The ultrasound features scores were significantly higher in upstream sides than in downstream sides(P=0.001).4.Histopathological analysis of the plaque:There was consistency between ultrasound features score and histopathologic analysis(Kappa=0.657,P=0.001).Histopathologic analysis showed there were more vulnerable plaque in the upstream sides than the downstream sides(P=0.021)and there were more lesions or ulcers in upstream sides than in downstream sides(P=0.046).There was consistency between ultrasound features parameter and histopathologic analysis.Conclusion1.There were significant differences of ultrasound features score,plaque thickness,stenosis rate between vulnerable plaque group and stable plaque group.2.The results of ultrasound features score and histopathologic analysis showed the vulnerability of upstream sides was higher than downstream side.Part Ⅱ The relation between carotid atherosclerotic plaques main tissue compositions distribution features and plaque vulnerabilityObjectiveTo explore the relation between carotid atherosclerotic plaques’ main tissue compositions distribution features and plaque vulnerability,and the difference in different parts of the plaques.Methods1.Research objects:The research subjects in this part are the same as the first part,the inclusion criteria are the same as the first part,and CD68 immunohistochemical staining,SMA staining,oil red O staining,and wolf scarlet-picric acid staining failure was added to the exclusion criteria 1-4 in the first part.A total of 51 patients(51 plaques)were included in the Part Ⅱ study,47 males and 4 females,mean age 63.7±6.9 years.2.Carotid artery ultrasound:The same as the first part.3.Histopathological features:Plaque HE staining process and pathological grouping criteria are the same as in the first part.The macrophages were dyeing by CD68 immunohistochemical staining,the smooth muscle cells by SMA staining,adipose tissue by oil red O staining,collagen tissue by Sirius scarlet picric acid staining.Taking pictures under the mirror,the software calculates the area ratio of each tissue component.And the plaque fiber cap features were observed.To research the distiction characteristics difference of macrophages,smooth muscle cells,adipose tissue,collagen and fibrous cap between vulnerable plaque group and stable plaque group,and also the difference of different parts of carotid atherosclerotic plaque(upstream side and downstream side).The correlation between the main tissue components within the plaque and the ultrasound feature score and the fiber cap was also researched.4.Statistical methods:Macrophages,smooth muscle cells,adipose tissue,collagen area ratios of different types,and fiber cap thickness in the vulnerable and stable plaque groups were analyzed using one-way ANOVA.Comparison of ultrasound feature scores between the two groups was performed using the Mann-Whitney U test.Intrapatomas,smooth muscle cells,adipose tissue,different types of collagen area ratios,and fiber cap thickness on the upstream and downstream sides of the plaque were tested by paired sample t test.The correlation of the area ratio of the main tissue components of carotid atherosclerotic plaque with ultrasonic characteristic parameters and fiber cap thickness was analyzed by Pearson correlation or Spearman grade correlation.Results1.Grouping:There were 51 cases in total,including 78.4%(40/51 cases)in vulnerable plaque group and 21.6%(11/51 cases)in stable plaque group.2.Macrophage distribution characteristics:The macrophage area ratios in vulnerable plaque group were significantly higher than those in stable plaque group(P=0.019).The macrophage area ratios in upstream side of plaques were significantly higher than those in downstream side(P=0.022).3.Smooth muscle cell distribution characteristics:The smooth muscle cell ratios in vulnerable plaque group were significantly lower than those in stable plaque group(P=0.009).The smooth muscle cell area ratios in upstream side of plaques were significantly lower than those in downstream side(P=0.012).4.Adipose tissue distribution characteristics:The adipose tissue area ratios in vulnerable plaque group were significantly higher than those in stable plaque group(P=0.001).The adipose tissue area ratios in upstream side of plaques were significantly higher than in downstream side(P=0.003).5.Collagen distribution characteristics:The collagen area ratios in vulnerable plaque group were significantly lower than those in stable plaque group(P=0.005).The collagen type Ⅰ area ratios in vulnerable plaque group were significantly lower than those in stable plaque group(P=0.033).The collagen type Ⅳ area ratios in vulnerable plaque group were significantly higher than those in stable plaque group(P=0.026).The collagen area ratios in upstream side of plaques were significantly lower than those in downstream side(P=0.032).The collagen type Ⅰ area ratios in upstream side of plaques were significantly lower than those in downstream side(P=0.022).The collagen type Ⅳ area ratios in upstream side of plaques were significantly higher than those in downstream side(P=0.016).6.Fibrous cap characteristes:The fibrous caps in vulnerable plaque group were significantly thinner than those in stable plaque group(P=0.012).The fibrous cap thicknesses in upstream side of plaques were significantly thinner than those in downstream side(P=0.001).7.Relevance studies:There was positive correlation between ultrasound features score and collagen type Ⅳ area ratio(P=0.036,correlation coefficient 0.83).There was positive correlation between fibrous cap thickness and collagen type Ⅰ area ratio(P=0.046,correlation coefficient 0.67).Conclusion1.Macrophage,adipose tissue,collagen type Ⅳ could increase the vulnerablity of carotid atherosclerotic plaque;Smooth muscle cell,collagen and collagen type I could increase the stability.2.The higher vulnerability of the upstream side of the plaque is related to the distribution features of macrophage,adipose tissue,smooth muscle cell,collagen and fibrous cap.Part Ⅲ The relation between carotid atherosclerotic plaques neovascular distribution characteristics and plaque vulnerabilityObjectiveTo evaluate the relation between carotid atherosclerotic plaques neovascular distribution characteristics and plaque vulnerability,and the difference of distribution in different parts of the plaque.Methods1.Research objects:The research subjects in this part are the same as the first part,the inclusion criteria are the same as the first part,and CD31 immunohistochemical staining failure was added to the exclusion criteria 1-4 in the first part.A total of 52 patients(52 plaques)were included in the Part Ⅲ research,47 males and 5 females,mean age 64.1±7.1 years.2.Carotid artery ultrasound:The same as the first part.3.Histopathological features:Plaque HE staining process,different parts of the plaque(upstream side,downstream side)treatment and pathological grouping criteria(vulnerable plaque group and stable plaque group)are the same as in the first part.Besides,all the plaques were also grouped into hemorrhage group or non-hemorrhage group,according to the presence of hemorrhage in plaque.The neovascular was dyeing by CD31 immunohistochemical staining.The difference of neovascular distribution characteristics(neovascular density,neovascular area ratio and neovascular diameter)was research between vulnerable plaque group and stable plaque group,also between hemorrhage group or non-hemorrhage group.And the difference between different sides of plaque was also researched.4.Statistical methods:For inter-group comparisons(vulnerable plaque group/stable plaque group or hemorrhage group/non-hemorrhage group),the difference in neovascular density,neovascular area ratio,and maximum neovascular diameter was analyzed using oneway ANOVA.The subject working characteristic curve(ROC)was used to assess the ability of neovascular distribution characteristics in diagnosing vulnerable plaques and in diagnosing intraplaque hemorrhage.The neovascular density,neovascular area ratio,and maximum neovascular diameter of the upstream and downstream sides of the plaque were compared by the paired sample t test.Results1.Grouping:There were 52 patients,including 76.9%(40/52 cases)in the vulnerable plaque group,23.1%(12/52 cases)cases in the stable plaque group,55.8%(29/52 cases)in the hemorrhage group,and 44.2%(23/52 cases)in the non-hemorrhage group.2.Neovascular distribution characteristics and plaque vulnerability:The neovascular density in vulnerable plaque group was significantly higher than in stable plaque group(P=0.031).The neovascular area ratio in vulnerable plaque group was significantly higher than in stable plaque group(P=0.008).The neovascular diameter in vulnerable plaque group was significantly higher than in stable plaque group(P=0.035).ROC curve analysis:neovascular density’ s ability to distinguish vulnerable plaque and stable plaque AUC 0.804,cut-off point 34.4;neovascular area ratio AUC 0.875,cut-off point 26.9%;neovascular diameter AUC 0.827,cut-off point 44.8μm.3.Neovascular distribution characteristics and intraplastic hemorrhage:The neovascular density in hemorrhage group was significantly higher than in non-hemorrhage plaque group(P=0.042).The neovascular area ratio in hemorrhage group was significantly higher than in non-hemorrhage plaque group(P=0.037).The neovascular diameter in hemorrhage group was significantly higher than in non-hemorrhage group(P=0.032).ROC curve analysis:neovascular density’s ability to distinguish hemorrhage group and nonhemorrhage group AUC 0.825,cut-off point 39.2;neovascular area ratio AUC 0.904,cut-off point 30.6%;neovascular diameter AUC 0.809,cut-off point 51.3μm.4.Neovascular distribution features in different parts of the plaque:The neovascular density in upstream side of plaques was significantly higher than in downstream side(P=0.042).The neovascular area ratio in upstream side of plaques was significantly higher than in downstream side(P=0.012).The neovascular diameter in upstream side of plaques was significantly bigger than in downstream side(P=0.028).Conclusion1.The augment of neovascular density,neovascular area ratio and neovascular diameter could increase carotid atherosclerotic plaque’s vulnerability and intraplastic hemorrhage significantly.2.Higher vulnerablity on the upstream side of plaque may be related to the higher neovascular density,neovascular area ratio and neovascular diameter.