Contrast-enhanced ultrasound perfusion patterns and serum lipid signatures of vulnerable carotid artery plaque in predicting stroke: A cohort study of carotid stenosis in Chinese patients

Abstract

BACKGROUND:

Early identification of vulnerable plaques at risk of rupture could help prevent cerebral ischemic stroke in patients with carotid artery disease.

OBJECTIVE:

To investigate the correlation between contrast-enhanced ultrasound (CEUS) perfusion patterns and serum lipid signatures of carotid artery plaques with the degree of carotid stenosis.

METHODS:

A total of 80 patients with carotid artery plaques who underwent CEUS were included. All patients underwent CEUS, computed tomography angiography or digital subtraction angiography, and serum lipid testing.

RESULTS:

The contrast agent enhancement levels and the CEUS perfusion patterns in the plaques were associated with the degree of carotid stenosis (P < 0.05). Serum free fatty acid (FFA) was associated with the contrast agent enhancement levels (P < 0.05), but did not correlate with the degree of stenosis (P > 0.05). There was no significant difference in total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides with respect to contrast agent enhancement levels (P > 0.05) or the degree of stenosis (P > 0.05).

CONCLUSION:

A high level of CEUS perfusion and increased serum FFA levels are indicative of vulnerable carotid plaques, which may be useful for the prediction of stroke in patients with carotid artery disease.

Keywords

Carotid plaque contrast-enhanced ultrasound perfusion patterns stenosis free fatty acid stroke

1 Introduction

Cardiovascular and cerebrovascular diseases were the leading cause of death worldwide in 2016. In particular, stroke remains the primary cause of death and disability among the adult population in China [1]. Therefore, it is important to reduce the incidence of stroke and to identify the patients most at risk [2].

Atherosclerotic carotid artery stenosis is thought to cause approximately 34% of ischemic strokes, with one-third of patients having more than moderate carotid stenosis (≥50%) [3]. Indeed, both extracranial carotid artery stenosis and severe intracranial carotid artery (ICA) stenosis are suggested to be predictive factors of stroke [4]. Ultrasound (US) has always been used as a first-line examination method in carotid system diseases, as it is non-invasive, inexpensive, and diagnostically accurate. US not only accurately determines the degree and scope of carotid stenosis, but also determines the shape and nature of the plaque, providing important information for clinical diagnosis and treatment options [5].

In addition to the degree of carotid stenosis, the vulnerability of carotid plaques is also an important risk factor for cerebrovascular accidents (CVAs) including stroke [9]. Both the tissue composition within the plaque and the hemodynamic environment outside the plaque are indicative of plaque vulnerability and rupture. In particular, the increased blood flow velocity resulting from the carotid stenosis may cause an increase in local shear stress within the plaque [6]. Although the early identification of plaques at risk of rupture could be useful for preventing CVA events, there is currently no standard clinical method to detect these vulnerable plaques.

Some studies [7] show it is not reliable to evaluate the stability of atherosclerotic plaques by conventional US alone. In addition, although digital subtraction angiography (DSA) remains the gold standard for the diagnosis of carotid artery stenosis, its application is limited due to its inability to evaluate the wall of the tube and the internal condition of the plaque, as well as its invasive nature, high cost, and large amount of radiation [8]. Meanwhile, computed tomography angiography (CTA) and high resolution magnetic resonance imaging (HRMRI) can accurately measure lumen stenosis and judge the nature of plaque; however, CTA cannot be used to evaluate plaque stability and HRMRI has several inherent limitations (e.g. long scanning time) [9]. Therefore, a novel method of accurately detecting plaque vulnerability in patients with carotid artery disease is required.

There is increasing evidence that intra-plaque neovascularization is significantly associated with plaque vulnerability [10, 11]. Intra-plaque neovascularization is currently considered to be the most powerful independent predictor of plaque rupture and hemorrhage, which are significantly correlated with the occurrence of clinical cardiovascular events [7 , 13]. Contrast-enhanced US (CEUS) is a new non-invasive technique that can clearly show the spatial location of blood vessels. A meta-analysis showed CEUS is a useful, non-invasive imaging modality to diagnose intra-plaque neovascularization, and thus can be used to predict plaque vulnerability [14, 15]. Therefore, CEUS may be a useful tool for detecting plaques at risk of rupture, for the early detection of cerebral ischemic stroke.

In this cohort study, we aimed to investigate the correlation among the CEUS perfusion pattern, serum lipid signatures and the degree of stenosis in patients with carotid plaques.

2 Materials and methods

2.1 Study subjects

This study was a retrospective, single-center study. Patients with carotid artery plaque were screened for routine US at the Tongren Hospital, Shanghai Jiaotong University School of Medicine. From March to June 2018, we screened 2039 patients with carotid plaques by routine US of the carotid arteries. We asked participants to complete questionnaires to obtain information about demographics, smoking history, and atrial fibrillation history. Physical examination was performed prior to the US examination, including the measurements of body mass index (BMI) and blood pressure. The study protocol was approved by the Institutional Ethics Review Committee(Shanghai Tongren Hospital 2018-030) and written informed consent was obtained from all participants.

A total of 1837 patients were excluded based on the following criteria: age <18 or >85 years, plaque thickness <1.5 mm, patients disagreed with the CEUS examination, and a history of myocardial infarction (MI) within two weeks. Among the 202 enrolled patients, 122 patients didn’t undergo CTA or DSA within a week of CEUS and with poor image quality. Finally, 80 patients that completed the CEUS baseline study were analyzed (Fig. 1).

Fig.1

Patient selection.

Complete information about demographics, smoking history, atrial fibrillation history, BMI, and blood pressure was obtained for all patients. In addition, all patients underwent CEUS, CTA or DSA, and serum lipid testing including total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, serum free fatty acid (FFA), among others.

2.2 Baseline examination and laboratory tests

In face-to-face interviews with participants, trained investigators used standard questionnaires to obtain information about their demographics, lifestyle, and history of diseases. Regular consumption of cigarettes in the past 6 months was defined as a current smoker. Blood samples were collected in the early morning after 10 h fasting for serum lipid testing. Participants’ body weight and height were obtained in light clothes and bare feet to the nearest 0.1 kg and 0.1 cm, respectively. BMI was calculated as the weight (kg) divided by the square of the height (m). Blood pressure was measured on the non-dominant arm in a seated position by an automated electronic sphygmomanometer three times consecutively with 1 min intervals, after at least 10 min rest. Hypertension and diabetes mellitus were determined according to the 2013 Hypertension Clinical Practice Guidelines [16] and the 2017 Standards of Medical Care in Diabetes [17], respectively.

2.3 Carotid US and CEUS imaging

All patients underwent bilateral duplex US evaluation of carotid arteries. To identify atherosclerotic lesions, a transverse sweep was recorded from the lower neck to the common carotid artery bifurcation to the external carotid artery and internal carotid artery. Then, the contrast agent (sulphur hexaflouride microbubbles, Bracco Altana Pharma, Germany) was injected into the peripheral vein for CEUS analysis. The contrast agent was prepared by diluting the powder in 5 mL sterile 0.9% NaCl solution. A 1.2 mL bolus of contrast agent was injected prior to flushing with 5 mL saline. Standard US evaluation and CEUS were performed by the same expert sonographer using the same dedicated ultrasonographic equipment (SIEMENS ACUSON OXANA 2) with an 8-MHz linear probe (9L4). The image parameters were set (by two US diagnosticians with >5 years experience) to show the plaque and surrounding tissue at the carotid stenosis as clear as possible. Carotid plaque neovascularization was assessed using CEUS.

Written informed consents of contrast examination were obtained from all participants. Exclusion criteria of CEUS were as follows: known allergy to albumin or the US contrast agent, with history of carotid endarterectomy and carotid stent, acute MI, connective tissue disease, malignancy, chronic kidney disease characterized by creatinine clearance lower than 30 mL/min and inadequate image acquisition to evaluate intra-plaque neovascularization on CEUS.

Based on the degree of enhancement of the carotid plaques shown on US, patients were classified as grade I to IV. In addition, the patients were divided into two groups according to their pattern of CEUS perfusion, i.e., whether the microbubbles of the contrast agent entered the plaque from the base or the surface (Fig. 2).

Fig.2

Examples of the two different types of plaque perfusion patterns observed on contrast-enhanced ultrasound (CEUS).

2.4 Measurement of extracranial internal carotid artery stenosis

Carotid artery stenosis was determined by CTA or DSA. According to the North American Symptomatic Carotid Endarterectomy Trial collaborators (NASCET), carotid artery stenosis can be classified as mild (i.e., arterial diameter is reduced less than 30%), moderate (i.e., arterial diameter is reduced by 30%–69%), severe (i.e., arterial diameter is reduced by 70%–99%), or occluded (i.e., no blood flow in the artery). In our study, patients were separated into three groups based on their degree of carotid artery stenosis as follows: mild (arterial diameter reduced <30%), moderate (arterial diameter reduced 30%–69%), and severe (arterial diameter reduced 70%–100%).

2.5 Cerebrovascular accident (CVA) event adjudication

CVA include stroke, transient ischemic attack, and stroke deaths that occur within half a year. Stroke was defined as a neurological deficit lasting at least 24 h or until death and accompanied by a brain imaging finding associated with stroke [2]. A transient ischemic attack was defined as a neurological deficit lasting between 30 s and 24 h but not accompanied by a brain imaging finding associated with stroke.

2.6 Statistical analysis

Continuous data is presented as mean±standard deviation (SD) or as median (interquartile range), as appropriate (skewed distribution). Continuous normally distributed variables were compared using the Student’s t test for independent samples and analysis of variance. Alternatively, the Mann-Whitney U test was used for independent samples and the Wilcoxon test for repeated measurements. The proportion of categorical variables was compared using a Chi-square test or the Fisher exact test. A P < 0.05 was considered to indicate statistical significance.

3 Results

3.1 CVA events and baseline characteristics

The relationship between CVA events and the baseline characteristics of the study participants is shown in Table 1. The mean age of patients was 70.19±11.28 years, and 71% of them were male. Overall, 62.5% of patients had hypertension, 48.8% had type 2 diabetes mellitus, 41.3% were current smokers, and 43.8% were overweight.

Table 1
Relationship between cerebrovascular accident (CVA) events and baseline characteristics of the study population (N = 80)

CVA (n = 58) Non-CVA (n = 22) All (n = 80) P-value

Age, y (mean±SD) 70.19±11.28 70.59±9.66 70.30±10.8

Sex (n, %) 0.233

Female 17 (29.3%) 9 (40.9%) 26 (32.5%)

Male 41 (71.7%) 13 (59.1%) 54 (67.5%)

Smoking status 0.213

Yes 26 (44.8%) 7 (31.8%) 33 (41.3%)

No 32 (55.2%) 15 (68.2%) 47 (58.7%)

Diabetes mellitus 0.456

Yes 29 (50%) 10 (45.5%) 39 (48.8%)

No 29 (50%) 12 (54.5%) 41 (51.2%)

Hypertension 0.555

Yes 36 (62.1%) 14 (63.6%) 50 (62.5%)

No 22 (37.9%) 8 (36.4%) 30 (37.5%)

Atrial fibrillation 0.473

Yes 6 (10.3%) 3 (13.6%) 9 (11.3%)

No 52 (89.7%) 19 (86.4%) 71 (88.7%)

Body mass index 0.142

Overweight (BMI ≥24) 28 (48.3%) 7 (31.8%) 35 (43.8%)

Normal (BMI <24) 30 (51.7%) 15 (68.2%) 45 (56.2%)

Total cholesterol 0.386

Abnormal (≥5.2 mmol/L) 17 (29.3%) 5 (22.7%) 22 (27.5%)

Normal (0∼5.2 mmol/L) 41 (70.7%) 17 (77.3%) 58 (72.5%)

HDL-C 0.394

Abnormal (≥1 mmol/L) 15 (25.9%) 7 (31.8%) 22 (27.5%)

Normal (0∼1 mmol/L) 43 (74.1%) 15 (68.2%) 58 (72.5%)

LDL-C 0.422

Abnormal (≥3.37 mmol/L) 11 (19.0%) 3 (81.0%) 14 (17.5%)

Normal (0∼3.37 mmol/L) 47 (81.0%) 19 (19.0%) 66 (82.5%)

Triglycerides 0.124

Abnormal (≥1.7 mmol/L) 20 (34.5%) 4 (18.2%) 24 (30%)

Normal (0∼1.7 mmol/L) 38 (65.5%) 18 (81.8%) 56 (70%)

Serum FFA 0.013

Abnormal (≥0.6 mmol/L) 21 (36.2%) 2 (9.1%) 23 (28.8%)

Normal (0∼0.6 mmol/L) 37 (63.8%) 20 (91.9%) 57 (71.2%)

Carotid artery stenosis (by CTA or DSA) 0.523

Stenosis 25 (43.1%) 10 (45.5%) 35(43.8%)

Non-stenosis 33 (56.9%) 12 (54.5%) 45(56.2%)

Contrast agent enhancement level 0.489

Grade I–II 31 (53.4%) 11 (50%) 42 (52.5%)

Grade III–IV 27 (46.6%) 11 (50%) 38 (47.5%)

CEUS perfusion patterns in plaque 0.606

Microbubbles infused from the 42 (72.4%) 16 (72.7%) 58 (72.5%)

surface of the plaque

Microbubbles infused from the 16 (27.6%) 6 (27.3%) 22 (27.5%)

base of the plaque

	CVA (n = 58)	Non-CVA (n = 22)	All (n = 80)	P-value
Age, y (mean±SD)	70.19±11.28	70.59±9.66	70.30±10.8
Sex (n, %)				0.233
Female	17 (29.3%)	9 (40.9%)	26 (32.5%)
Male	41 (71.7%)	13 (59.1%)	54 (67.5%)
Smoking status				0.213
Yes	26 (44.8%)	7 (31.8%)	33 (41.3%)
No	32 (55.2%)	15 (68.2%)	47 (58.7%)
Diabetes mellitus				0.456
Yes	29 (50%)	10 (45.5%)	39 (48.8%)
No	29 (50%)	12 (54.5%)	41 (51.2%)
Hypertension				0.555
Yes	36 (62.1%)	14 (63.6%)	50 (62.5%)
No	22 (37.9%)	8 (36.4%)	30 (37.5%)
Atrial fibrillation				0.473
Yes	6 (10.3%)	3 (13.6%)	9 (11.3%)
No	52 (89.7%)	19 (86.4%)	71 (88.7%)
Body mass index				0.142
Overweight (BMI ≥24)	28 (48.3%)	7 (31.8%)	35 (43.8%)
Normal (BMI <24)	30 (51.7%)	15 (68.2%)	45 (56.2%)
Total cholesterol				0.386
Abnormal (≥5.2 mmol/L)	17 (29.3%)	5 (22.7%)	22 (27.5%)
Normal (0∼5.2 mmol/L)	41 (70.7%)	17 (77.3%)	58 (72.5%)
HDL-C				0.394
Abnormal (≥1 mmol/L)	15 (25.9%)	7 (31.8%)	22 (27.5%)
Normal (0∼1 mmol/L)	43 (74.1%)	15 (68.2%)	58 (72.5%)
LDL-C				0.422
Abnormal (≥3.37 mmol/L)	11 (19.0%)	3 (81.0%)	14 (17.5%)
Normal (0∼3.37 mmol/L)	47 (81.0%)	19 (19.0%)	66 (82.5%)
Triglycerides				0.124
Abnormal (≥1.7 mmol/L)	20 (34.5%)	4 (18.2%)	24 (30%)
Normal (0∼1.7 mmol/L)	38 (65.5%)	18 (81.8%)	56 (70%)
Serum FFA				0.013
Abnormal (≥0.6 mmol/L)	21 (36.2%)	2 (9.1%)	23 (28.8%)
Normal (0∼0.6 mmol/L)	37 (63.8%)	20 (91.9%)	57 (71.2%)
Carotid artery stenosis (by CTA or DSA)				0.523
Stenosis	25 (43.1%)	10 (45.5%)	35(43.8%)
Non-stenosis	33 (56.9%)	12 (54.5%)	45(56.2%)
Contrast agent enhancement level				0.489
Grade I–II	31 (53.4%)	11 (50%)	42 (52.5%)
Grade III–IV	27 (46.6%)	11 (50%)	38 (47.5%)
CEUS perfusion patterns in plaque				0.606
Microbubbles infused from the	42 (72.4%)	16 (72.7%)	58 (72.5%)
surface of the plaque
Microbubbles infused from the	16 (27.6%)	6 (27.3%)	22 (27.5%)
base of the plaque

All data are given as n (%), unless otherwise indicated.

Serum FFA was significantly associated with CVA events (P < 0.05). No significant association was found between CVA events and patients’ demographics, smoking history, atrial fibrillation history, BMI, diabetes mellitus, hypertension, total cholesterol, HDL-C, LDL-C, or triglyceride levels. In addition, no significant association was found between CVA events and the degree of carotid artery stenosis, the contrast agent enhancement levels, or the CEUS perfusion patterns in the plaques (P > 0.05; Table 1).

3.2 Degree of stenosis and the enhancement level of the plaques

There were 35 patients in the carotid stenosis group (27 cases of mild stenosis, three cases of moderate stenosis, and five cases of severe stenosis), and 45 patients in the non-stenosis group. Based on the enhancement level of the plaques, patients were given one of four grades (Table 2). There were no patients classified as grade I (no appearance of enhancement within the plaque), 45 were classed as grade II (limited appearance of enhancement within the plaque), 25 patients as grade III (less enhancement than that noted in grade IV and more than that of grade II), and 10 patients as grade IV (presence of a pulsating arterial vessel within the plaque). We found that as the severity of carotid artery stenosis increased, the degree of contrast agent perfusion also increased (Fig. 3).

Table 2
Relationship between the enhancement level of the plaques and the degree of stenosis

Enhancement level of the plaques

Degree of stenosis Total Grade I Grade II Grade III Grade IV

Non-stenosis 45 0 32 10 3

Mild 27 0 10 11 6

Moderate 3 0 2 1 0

Severe 5 0 1 3 1

	Enhancement level of the plaques
Non-stenosis	45	32	10	3
Mild	27	10	11	6
Moderate	3	2	1	0
Severe	5	1	3	1

Fig.3

The relationship between the enhancement level of the plaques (grade I to IV) and the degree of stenosis.

3.3 Degree of stenosis and CEUS perfusion patterns

In 52 cases, the contrast agent used for CEUS entered the interior of the plaque from the base and in 28 cases, it infused from the surface (Table 3). In the non-stenosis group, seven CEUS images (15.6%) were characterized by surface infusion compared to 21 cases (60%) in the stenosis group (Fig. 4). The contrast agent enhancement levels and the CEUS perfusion patterns in the plaques were significantly associated with the degree of carotid stenosis (P < 0.05). It was shown that the plaque surface entering mode of the stenosis group was more than the non-stenosis group. Most of the plaque showed surface entering mode often combined with base entering mode.

Table 3
Relationship between contrast-enhanced ultrasound (CEUS) perfusion pattern and degree of stenosis

CEUS perfusion patterns

Degree of stenosis Microbubbles infused from the surface to the interior of the plaque Microbubbles infused from the base to the interior of the plaque Total P-value

Non-stenosis 7 38 45 <0.05

Stenosis 21 14 35

	CEUS perfusion patterns
Non-stenosis	7	38	45	<0.05
Stenosis	21	14	35

Fig.4

The relationship between the contrast-enhanced ultrasound (CEUS) perfusion pattern and the degree of stenosis.

3.4 Correlation between serum lipid levels, CEUS perfusion levels, and the degree of stenosis

There was a positive correlation between serum FFA concentration and contrast agent perfusion level in the plaque (P < 0.05; Table 4), but no significant correlation with the degree of stenosis. There was no significant difference in the serum lipid testing index (including total cholesterol, HDL-C, LDL-C, triglycerides, and serum FFA) between the stenosis group and the non-stenosis group (P > 0.05).

Table 4
Relationship between free fatty acid (FFA) levels and contrast-enhanced ultrasound (CEUS) perfusion levels

CEUS perfusion level

Total Grades I–II Grades III–IV P-value

FFA within normal range 57 26 31 <0.05

FFA out of normal range 23 19 4

	CEUS perfusion level
FFA within normal range	57	26	31	<0.05
FFA out of normal range	23	19	4

4 Discussion

Neovascularization and intra-plaque hemorrhage are necessary steps to transform stable plaques into unstable plaques, eventually leading to plaque rupture [3]. Many clinical and animal models have confirmed there is a close relationship between the degree of neovascularization and intra-plaque hemorrhage [10 –12]. In the guidelines for the clinical application of CEUS in non-liver clinics, the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) recommended grade B;1b for the evaluation carotid plaques neovascularization in 2011. The guidelines highlight that CEUS can show the formation of vascular plaque ulcers, and correlates well with the results of histological examination [18]. CEUS can also provide more information about plaque stability than conventional US evaluation [19].

We did not find a correlation between the extent of plaque perfusion, perfusion patterns, and CVA events in our cohort of patients with carotid artery stenosis; however, this may have been because the CVAs were not caused by the particular plaques we examined. Meanwhile, we found significant differences between patients with and without stenosis in terms of their contrast enhancement level on CEUS, indicating there was more neovascularization present in those with stenosis. We also found significantly different patterns of plaque entry between patients with and without stenosis. In patients with stenosis, the contrast agent appears to enter or perfuse via the plaque surface rather than from the base of the plaque. This is likely because the local shear force of the plaque is increased in those with stenosis, causing the surface of the plaque to crack and become vulnerable. Although there was no obvious defect on the conventional ultrasound image at the perfusion entrance of the plaque surface (Fig. 2), we did observe the contrast agent entering the plaque from the vascular lumen on CEUS during the regression stage. Therefore, the mode of plaque entry of the contrast agent used for CEUS may provide early clues to vulnerable changes in plaques.

In our study, the difference in FFA concentration and intraplaque CEUS was statistically significant in patients with CVAs. Previous studies have shown FFA is closely related to lipid metabolism [20]. It has also been suggested that when the concentration of FFA in the blood is too high, micelles and fatty acid vesicles with an acidic core may form, which fuse with endothelial cells and initiate plaque formation [25]. As an independent predictor of death from cardiovascular and cerebrovascular diseases, FFA could be pathogenically involved in the atherosclerotic and stroke process [21, 22]. Many studies have shown an increase in FFA concentration is closely related to stroke events. In particular, elevated blood FFA concentration has been shown to increase the risk of cardiovascular disease events [25], which is similar to our results [23, 24].

5 Conclusion

As the degree of carotid artery stenosis increases, so too does the level of CEUS perfusion in the carotid plaques. We showed the contrast agent mainly infuses from the surface to the interior of the plaque on CEUS in those with moderate or higher carotid stenosis plaques. Furthermore, the enhancement level of the plaques on CEUS increased with increasing FFA concentration. Together these factors may provide evidence of vulnerable plaques in those with carotid artery stenosis.

6 Study limitations

First, this is a retrospective study, with all its inherent limitations. In particular, as the carotid plaque images were only obtained at a single time point, we could not determine how the carotid artery measurements change over time, nor can we prove a causal relationship. Second, this study is limited because of its small sample size. Third, we only examined two-dimensional images in all our examinations. Three-dimensional imaging can provide information on the whole plaque, so it has absolute advantages in assessing the stability of carotid plaques [25]. However, the two-dimensional image has only a single “most representative” section to represent the plaque. Because of the uneven neovascularization distribution in plaque, two-dimensional CEUS angiography has limitations.

Finally, we only assessed the neovascularization of plaques qualitatively, via direct observation. Although this method may seem subjective, it has good repeatability. At present, quantitative evaluation of CEUS images is more challenging than qualitative evaluation in clinical applications, and studies indicate such quantitative methods may not have any added benefit over the qualitative methods [26, 27]. Therefore, qualitative methods of CEUS remain the gold standard in this field [14], with standardized quantifiable CEUS methods requiring further exploration in clinical applications.

Conflicts of interest

The authors declare that they have no competing interests.

Footnotes

Acknowledgments

The authors are grateful for the medical staff of the Department of Medical Ultrasound, Tongren Hospital, Shanghai Jiaotong University School of Medicine.

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	Enhancement level of the plaques
Degree of stenosis	Total	Grade I	Grade II	Grade III	Grade IV
Non-stenosis	45	0	32	10	3
Mild	27	0	10	11	6
Moderate	3	0	2	1	0
Severe	5	0	1	3	1

	CEUS perfusion patterns
Degree of stenosis	Microbubbles infused from the surface to the interior of the plaque	Microbubbles infused from the base to the interior of the plaque	Total	P-value
Non-stenosis	7	38	45	<0.05
Stenosis	21	14	35

	CEUS perfusion level
	Total	Grades I–II	Grades III–IV	P-value
FFA within normal range	57	26	31	<0.05
FFA out of normal range	23	19	4