Contrast-enhanced Ultrasound Imaging Of Atherosclerotic Plaque Neovascularization: Animal Experiments And Clinical Assessment

Part one:Animal ExperimentsChapter 1 Establishment and evaluation of atherosclerosis rabbit modelObjective:To establish a rabbit model of atherosclerosis (AS) by feeding hypercholesterol diet. Methods:Twelve healthy New Zealand rabbits weighed 2.0±0.2kg were fed with hypercholesterol diet. Before and after the feeding with hypercholesterol diet, ultrasound examination was performed to observe the changes of abdominal aortic wall. The abdominal aortas were observed by pathology to verify the atheroselerotic lesion. Results:The AS model was successfully established in 9 rabbits. The weight of the rabbits was significantly increased after the feeding with hypercholesterol diet (4.3±0.5kg vs 2.1±0.1kg, P?0.0001). The typical carotid AS plaques which contained fibrous cap, calcification and lipid core area were observed pathologically. Conclusion:The experimental plaques presented great comparability to human AS plaques. Therefore, it is feasible to prepare a AS model induced by feeding hypercholesterol diet in rabbits.Chapter 2 Quantitative analysis of plaque neovascularization with contrast-enhanced ultrasound in rabbit model:a pilot studyObjective:To verify contrast-enhanced ultrasound (CEUS) quantitative analysis of plaque enhancement is able to assess the intraplaque neovascularization. Methods: The abdominal aortas were examined with CEUS in 9 AS rabbits established by feeding high-fat diet. Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. Contrast agent SonoVue was used with a dose of 0.2ml. The enhanced intensity (EI) of the plaque and the ratio of El within the plaque to that in the lumen of the carotid artery was calculated with QLAB software. Each aorta specimen was stained with F8 (von willebrand factor) for microvessel tagging. The quantitative parameters were compared with the immunohistochemical study results. Results:The mean value of El and ratio were 3.91±0.87dB and 0.30±0.06, respectively. Five specimens showed positive F8 staining and 4 showed negative. El and ratio were significantly greater in positive group than in negative group (4.47±0.60 dB vs 3.22±0.62dB,0.34±0.05vs0.25±0.05). CEUS quantitative parameters El (r=0.779, P=0.013) and ratio (r=0.693, P=0.019) correlated to microvessels marked with F8, respectively. Conclusions:It is histologically validated that CEUS quantifies the neovascularization in AS plaques. This imaging technique provides a window for plaque risk stratification.Part two:Clinical AssessmentChapter 1 Quantitative contrast-enhanced ultrasound analysis of carotid plaque neovascularization in relation to semiquantitative analysisObjective:To analyze the relation between the degree of carotid plaque enhancement visualized by CEUS and quantitative parameters of enhancement intensity in the plaques. Methods:Conventional and contrast-enhanced ultrasound were performed on 135 patients (119 males,16 females, mean age,69.1years±8.3) in which bilateral atherosclerotic lesions were detected in 32 patients. Therefore, a total of 167 carotid arteries and plaques were documented and analyzed. Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. Contrast agent SonoVue was used. Enhancement within the plaque was categorized as grade 0 to grade 4 by visual interpretation and was evaluated by quantitative analysis. The enhanced intensity (El) of the plaque and the ratio of El within the plaque to that in the lumen of the carotid artery were calculated. Results: There were 54 plaques classified as grade 0 on CEUS,18 plaques as grade 1,45 as grade2,25 as grade 3 and 25 as grade 4. Fourteen plaques (14/167,8.4%) were not available for CEUS quantitative analysis due to the calcified plaques attenuation. El and ratio were significantly different between plaques of different CEUS enhancement grades (P?0.0001). El and ratio were not significantly different between groups of similar CEUS grades. Conclusion:The degree of carotid intraplaque enhancement evaluated with quantitative analysis correlated with that with semiquantitative assessment. The quantitative and semiquantitative methods provide us different choice to meet different clinical purpose.Chapter2 Correlation of carotid artery IMT with intraplaque neovacularization detected with contrast-enhanced ultrasoundObjective:To analyze the relation between common carotid artery (CCA) intima-media thickness (IMT) and the degree of carotid plaque enhancement on CEUS. Methods:Conventional and contrast-enhanced ultrasound were performed on 135 patients (119 males,16 females, mean age,69.1years±8.3) in which bilateral atherosclerotic lesions were detected in 32 patients. Therefore, a total of 167 carotid arteries and plaques were documented and analyzed. Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. IMT of CCA was measured with QLAB software. Contrast-enhancement within the plaque was categorized as grade 0 to grade 4 by visual interpretation and was evaluated by quantitative analysis. The enhanced intensity (EI) of the plaque and the ratio of El within the plaque to that in the lumen of the carotid artery were calculated. Results:IMT was not measured on 6 carotid arteries. In 161 carotid arteries, IMT was not associated with the degree of intraplaque enhancement on CEUS (P=0.994). IMT was not associated with EI (P=0.687) and ratio (P=0.852), either. Conclusion:Carotid plaque enhancement was not associated with the IMT of CCA. Therefore, CEUS may be helpful in the monitoring and stratification of patients with carotid plaques and normal IMT as a new biomarker.Chapter 3 Correlation of carotid artery plaque echogenicity with intraplaque neovacularization detected with contrast-enhanced ultrasoundObjective:To correlate echogenicity of atherosclerotic carotid artery plaques at stansard ultrasound with the degree of intraplaque neovacularization detected with contrast-enhanced ultrasound. Methods:Conventional and contrast-enhanced ultrasound were performed on 135 patients (119 males,16 females, mean age, 69.1 years±8.3) in which bilateral atherosclerotic lesions were detected in 32 patients. Therefore, a total of 167 carotid arteries and plaques were documented and analyzed. Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. The contrast agent SonoVue was used. Plaque echogenicity was classified as hypo, iso, hyperechoic and heterogeneous. Contrast-enhancement within the plaque was categorized as grade 0 to grade 4 by visual interpretation and was evaluated by quantitative analysis. The enhanced intensity (EI) of the plaque and the ratio of El within the plaque to that in the lumen of the carotid artery were calculated. Results:In a total of 167 AS plaques, echogenicity was inversely correlated with grade of intraplaque neovasculaization (P<0.0001) detected with CEUS. More echolucent lesions had a higher degree of neovascularization compared with more echogenic ones. EI (P=0.021) and ratio (P=0.012) were also correlated with plaque echogenicity inversely. The degree of intraplaque enhancement, EI and ratio were significantly different between hypoechoic group and other groups. Conclusion:CEUS may provide more valuable information for risk stratification of carotid plaques than the echogenicity detection of conventional US. It may be helpful for screening high risk patients.Chapter 4 Correlation of severity of carotid artery stenosis with intraplaque neovacularization detected with contrast-enhanced ultrasoundObjective:To evaluate contrast-enhanced ultrasound imaging of carotid plaques with different stenostic degree as a clinical tool to study intraplaque neovascularization. Methods:Standard and contrast-enhanced ultrasound were performed on 92 patients (83 males,9 females,67.7years±8.5) suspected of carotid stenosis in which bilateral atherosclerotic lesions were detected in 22 patients. Therefore, a total of 114 carotid arteries and plaques were documented and analyzed. The degree of stenosis was determined by intravenous digital subtraction angiography (DSA). Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. The contrast agent SonoVue was used. Contrast-enhancement within the plaque was categorized as grade 0 to grade 4 by visual interpretation and was evaluated by quantitative analysis. The enhanced intensity (EI) of the plaque and the ratio of EI within the plaque to that in the lumen of the carotid artery were calculated. Results:In a total of 114 carotid arteries, artery occlusion was determined in 9 arteries,>90%stenosis was detected in 25 arteries, 70%-89%stenosis in 47,50%-69%stenosis in15 and<50% stenosis in 18. The degree of stenosis significantly correlated to grades of intraplaque enhancement (P<0.0001). In the group with stenosis less than 70%, stenosis degree was not associated with the grade of contrast enhancement (P=0.151). EI (P=0.864) and ratio (P=0.636) were not associated with the degree of stenosis. Conclusion:Carotid plaque contrast enhancement was not associated with the degree of artery stenosis. Therefore, CEUS may be a valuable tool for further risk stratification of carotid plaques with different degrees of carotid artery stenosis.Chapter 5 Contrast enhancement of carotid plaques in relation to clinical ischemic symptomsObjective:To determine the correlation between the degree of carotid plaque enhancement with ultrasound contrast agent and clinical ischemic symptoms in patients with carotid atherosclerosis. Methods:One hundred and thirty-five patients (119 males,16 females, mean age,69.1 years±8.3) with carotid plaques were studied with standard and contrast-enhanced ultrasound. Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. The contrast agent SonoVue was used. Contrast-enhancement within the plaque was categorized as grade 0 to grade 4 by visual interpretation and was evaluated by quantitative analysis. The enhanced intensity (EI) of the plaque and the ratio of EI within the plaque to that in the lumen of the carotid artery were calculated. According to clinical symptoms, all patients were classified into 3 groups: (1) with acute ischemic stroke; (2) symptomatic; (3) asymptomatic. Results: Contrast enhancement of the carotid plaques was correlated to the clinical symptom (P= 0.0476). In the former two groups grade 2-4 enhancement within the plaque was significantly greater than those in the asymptomatic group (P=0.0061). EI and ratio showed no significant difference between 3 groups. Conclusion:CECU imaging may provide a noninvasive adjunctive method to risk stratify individuals by identifying intraplaque neovascularization and may serve as a valuable screening tool to identify patients at high risk of cerebrovascular events.Chapter 6Quantification of carotid plaque neovascularization using contrast ultrasound:a histologic validationObjective:To evaluate the carotid plaque neovascularization with quantitative analysis of contrast-enhanced ultrasound images and to compare the results with the histopathologic analysis of the plaque. Methods:Seventeen patients (16 males,1 female) with 17 carotid plaques were studied with standard and contrast-enhanced ultrasound. Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software. The contrast agent SonoVue was used with a dose of 2.0ml. Contrast-enhancement within the plaque was categorized as grade 0 to grade 4 by visual interpretation and was also evaluated by quantitative analysis with software. The enhanced intensity (El) of the plaque and the ratio of El within the plaque to that in the lumen of the carotid artery were calculated. The surgical specimen was available for histological determination of the cap thickness and the lipid core size of the plaques. Each specimen was stained with CD34 for microvessel, CD68 for macrophages and VEGF. Results: Contrast-enhancement within the plaque was associated with neovascularization at histology. Plaques with higher contrast enhancement showed greater neovascularization at histology. Contrast-enhancement in the plaque was not associated with the cap thickness, lipid core size, inflammation infiltration (macrophages) and VEGF determined by histological examination. The echogenicity of plaques was not associated with those histological features, neither. Conclusion: Carotid plaque contrast enhancement correlates to the neovessel density in the plaque. Contrast-enhanced ultrasound imaging may provide valuable information for plaque risk stratification as a noninvasive and simple method.