High Resolution Vessel Wall Image With Macrophage-target Contrast Agent To Identify Early Atherosclerotic Plaques And Explore Its Progression Characteristics

Objective:To qualitative and quantitative analysis of the morphology,pathology,and signal change of early plaques in rabbit carotid atherosclerotic model during different periods,with the pathological results as the gold standard by the application of high-resolution magnetic resonance angiography（HR-VWI）combined with macrophage targeted contrast agent.Materials and Methods:Part Ⅰ:Fifty New Zealand rabbits were randomly divided into experimental group（n=46）and control group（n=4）.The carotid artery atherosclerosis model was established by carotid artery balloon injury combined with a high-fat diet.HR-VWI and Gd modified macrophage targeted gold nanoclusters（Gd MG NCs）were combined to enhance the plaque at each time point（4th,8th,12th,16th,20th week）.The images were analyzed by using Radi Ant Dicom Viewer.The number of plaques,the area of the vessel wall(Area_wall),and the plaque area(Area_plaque)were recorded.The number of macrophages with positive expression of scavenger receptor group a type I（SR-AI）and the number of macrophages with positive expression of CD68 antibody（CD68 density）were calculated.The changes in HR-VWI measurements and pathological indexes were analyzed at different time points.The differences between the results of targeted and non targeted enhanced HR-VWI were compared.The correlation between the number of plaque detected by targeted enhanced HR-VWI,areawall,areaplaque and the density of SR-AI and CD68 was analyzed.Part Ⅱ:Image J software was used to delineate the lipid core area and wall area of plaque on non-enhanced and enhanced HR-VWI images to measure the image vulnerability index(VI_VWI).According to the classification of plaque by AHA,the VI_VWIof plaque was divided into 0～4 grades:no lipid,VI_VWI=0;lipid content<5%,VI_VWI=1;5%≤lipid content<30%,VI_VWI=2;30%≤lipid content<40%,VI_VWI=3;lipid content≥40%,VI_VWI=4.The pathological sections were strictly matched with HR-VWI,and Image J was used to analyze lipid,collagen,muscle fiber,and macrophage contents.The pathological vulnerability index(VI_pathology)=（macrophage positive area%+lipid staining area%）/（smooth muscle cell staining area%+collagen fiber staining area%）was calculated,and the pathological vulnerability index(VI_pathology)was calculated.The relationship between HR-VWI and pathological vulnerability was compared.ROC curve was used to evaluate the diagnostic efficiency of HR-VWI for plaque vulnerability.Part Ⅲ:Image J was used to analyze the histogram of HR-VWI images of plaques from the rabbits which obtained all the HR-VWI examinations.The development features of plaque coefficient of variation（CV）,skewness,kurtosis,and standard deviation（SD）at each time point after balloon injury were observed.The histogram parameters of vulnerable plaques and stable plaques were compared with pathological results as the gold standard.ROC curve was used to analyze the diagnostic efficiency of histogram parameters for vulnerable plaque and explore the diagnostic value of histogram to diagnose vulnerable plaque.Results:Part Ⅰ:In pair comparison,the averaged detected plaque count of targeted-enhanced HR-VWI was statistically more than non-targeted enhancement at each time point（p>0.05）.In the targeted-enhanced group,the average detection showed an increase trend,the 4^th～8^thincrease 0.63±0.14（p=0.002）,the 8^th～12^thand12^th～16^thshowed no significantly different（p>0.05）,the 16^th～20^thincrease 0.68±0.15（p=0.003）.In the non-targeted-enhanced group,the average detection showed an increase trend,the 4^th～8^thincrease 0.47±0.14（p=0.034）,the 8^th～12^thshowed no significantly different（p>0.05）,and 12^th～16^thincrease 0.47±0.14（p=0.034）,the16^th～20^thincrease 0.37±0.11（p=0.045）.In the 4th and 8th week,the plaque area measured by targeted enhancement was larger than measured by non-targeted enhancement（p<0.001）.The plaque area measured by non-targeted enhancement was larger than measured by targeted enhancement from the 12th week,but the difference was not significant.SR-AI density values at the 8^th,12^th,and 16^thweeks after injury were significantly lower than those at the 20^th（p<0.05）.CD68 density at4^th～8^thweeks after injury were significantly increased（p=0.034）.The correlation analysis of HR-VWI measurement and pathological indexes at different time points after injury showed that plaque area was highly correlated with SR-AI density（r=﹣0.757,p<0.001）and CD68 density（r=﹣0.696,p<0.001）,while vessel wall area was not correlated with SR-AI density（r=﹣0.224,p=0.234）and CD68 density（r=﹣0.102,p=0.592）.Part Ⅱ:There’s no significant difference between plain scan and enhanced HR-VWI in evaluating plaque image vulnerability index（p=0.175）.There was no significant difference in measuring the plaque vulnerability index between the two groups at the 4th,8th,12th,and 16th week（p>0.05）.Only at the 20th week,VI_VWImeasured by plain scan was 1.500 smaller than measured by enhanced scan（p=0.014）.The vulnerability index of plaque image measured at each time point after injury increased and showed an upward trend.There was no significant difference between the baseline（4 weeks）.Simultaneously,there was no significant difference between the point before and after injury measured by enhanced HR-VWI（p>0.05）.The difference in image vulnerability index at the 20th week was statistically significant.The image vulnerability index at the 20th week increased by 3.75（p=0.006）compared with the 4th week and 2.75（p=0.016）compared with the 8th week.The sensitivity,specificity,and AUC of non-contrast HR-VWI were 90.00%,90.90%,and 90.00%,respectively;the sensitivity,specificity,and AUC of enhanced HR-VWI were 95.45%,100%,and 95.45%,respectively;there was no significant difference between enhanced and non-enhanced HR-VWI by Delong test（p=0.527）.Part Ⅲ:The Repeated Measures ANOVA was used to analyze the HR-VWI histograms of the lesions（n=21）with pathological results at the end of 20 weeks after the balloon injury.The CV of plaque at each time point after injury（4th,8th,12th,16th,20th week,the same below）was 2.77±0.14,3.26±0.38,3.33±0.30,4.02±0.53,3.78±0.08,respectively.There was a significant difference between the mean CV of each time point（p<0.001）.The CV of plaque at the 4th to 8th week after injury increased significantly（p=0.010）,and there was no significant difference between the other adjacent time points（p>0.05）.The CV at the 12th,16th,and 20th weeks after injury were significantly higher than that at the 4th weeks（p<0.05）,but there was no significant difference at other time points（p>0.05）.There was no significant difference in kurtosis,skewness,and SD of plaque at each time point after injury（p>0.05）.Furthermore,repeated measurement ANOVA was used to analyze the histogram parameters of vulnerable plaque（n=10）.The CV value of each adjacent time point after injury increased significantly between 4 and 8 weeks（p=0.044）,increased significantly between 12 and 16 weeks（p=0.040）,decreased significantly between 16 and 20 weeks（p=0.040）.There was no significant difference between 8 and 12 weeks（p=1.000）.There was no significant difference in CV between the other time points（p>0.05）.There was no significant difference in kurtosis,skewness,and SD of vulnerable plaque at each time point（p>0.05）.The CV of stable plaque（n=11）at each time point was 2.74±0.16,3.24±0.42,3.24±0.35,3.83±0.59,3.81±0.03,respectively.There was a significant difference between the 4th and 20th week（p=0.001）,but there was no significant difference among other time points（p>0.05）.There was no significant difference in kurtosis,skewness,and SD of stable plaques at each time point（p>0.026）.There was no interaction between CV,kurtosis,skewness,and SD（all p>0.05）,and there was no significant difference between the parameters of vulnerable and stable plaques at different time points（all p>0.05）.ROC curve was used to analyze the diagnostic efficiency of histogram parameters for plaque vulnerability.It was found that CV had the highest diagnostic efficiency for plaque vulnerability,AUC=88.5%,the best cut-off value=4.11,but there was no statistical difference in AUC of each parameter in the Delong test（p>0.05）.Conclusion:Firstly,this study combined HR-VWI and macrophage-specific targeted contrast agent to realize the early detection of atherosclerotic lesions in the rabbit carotid atherosclerotic plaque model.We analyzed the changing trend and difference of plaque detection number,vascular wall area,and plaque area measured by targeted-enhanced HR-VWI and non-targeted enhanced HR-VWI,exploring dynamic development characteristics of plaque,and compared targeted enhanced and non targeted enhanced HR-VWI.The difference was measured by non-targeted enhancement.By analyzing the correlation between the targeted-enhanced HR-VWI measurements and the pathological indexes of macrophages,it was found that the measured plaque area and vascular wall area were related to the density of CD68,which proved that the early infiltration of macrophages was related to the development of plaque.Secondly,the plaque vulnerability progression trend measured by plain HR-VWI and enhanced HR-VWI was analyzed and compared with pathological results.It is proved that HR-VWI can qualitatively and quantitatively evaluate the changing characteristics of plaque vulnerability progression.Finally,repeated measures ANOVA and multivariate ANOVA were used to analyze the histogram parameters of vulnerable plaque and stable plaque.It was found that the variation of coefficient of variation could reflect the development trend of vulnerable plaque,and the coefficient of variation had a high diagnostic effect on the diagnosis of plaque properties.