Pathological Quantification of Carotid Artery Plaque Instability in Patients Undergoing Carotid Endarterectomy

Abstract

Background: Unstable atherosclerotic carotid plaques cause cerebral thromboemboli and ischemic events. However, this instability has not been pathologically quantified, so we sought to quantify it in patients undergoing carotid endarterectomy (CEA).

Methods and Results: Carotid plaques were collected during CEA from 67 symptomatic and 15 asymptomatic patients between May 2015 and August 2016. The specimens were stained with hematoxylin-eosin and elastica-Masson. Immunohistochemistry was performed using an endothelial-specific antibody to CD31, CD34 and PDGFRβ. The histopathological characteristics of the plaques were studied. By multiple-variable logistic regression analysis, plaque instability correlated with the presence of plaque rupture [odds ratio (OR), 9.75; P=0.013], minimum fibrous cap thickness (OR per 10 μm 0.70; P=0.025), presence of microcalcifications in the fibrous cap (OR 7.82; P=0.022) and intraplaque microvessels (OR 1.91; P=0.043). Receiver-operating characteristics analyses showed that these factors combined into a single score diagnosed symptomatic carotid plaques in patients with carotid artery stenosis with a high level of accuracy (area under the curve 0.92; 95% confidence interval 0.85–0.99 vs. asymptomatic).

Conclusions: This analysis of carotid plaque instability strongly suggested that the diagnostic scoring of carotid plaque instability improves the understanding and treatment of carotid artery disease in patients undergoing CEA.

Ischemic strokes and transient ischemic attacks (TIA) are usually caused by unstable carotid lesions, which result in thrombus formation and occlusion of the artery. The histological characteristics of unstable carotid plaques have been described in several studies.^1–4 In studies of coronary arteries, unstable plaques have been identified as ruptured, inflammatory plaques with thin fibrous caps (TFC) that were often the cause of acute coronary syndromes rather than stable angina.^5,6 In the cerebral circulation, however, most ischemic strokes are caused by distal embolization originating from atherosclerotic plaques, or by an acute, instead of chronic, occlusion of a carotid artery.⁷ The interest in the morphology and functional characteristics of carotid plaques, using various imaging techniques and biochemical markers, has been growing.^8–13 The progression of carotid plaques accelerated by hemorrhages developing inside the plaque has been observed in several imaging studies, and has been associated with predictors of future ischemic cerebrovascular events (CVE).^14,15 In previously asymptomatic patients presenting with 50–79% carotid artery stenoses, thin or ruptured fibrous caps, hemorrhages within a plaque, and large, lipid-rich and necrotic cores (NC) have been associated with the development of adverse CVE.¹⁶ However, there is no comprehensive, pathological measure of carotid plaque instability that can be used to predict the risk of ischemic CVE. We performed this study in patients who underwent carotid endarterectomy (CEA) to describe in detail the pathology of carotid atherosclerosis and develop a diagnostic pathological scoring of unstable plaques.

Methods

Sample Population

We analyzed data from 74 men and 8 women aged >30 years who, between May 2015 and August 2016, consecutively underwent CEA in the departments of neurosurgery of Nakamura Memorial Hospital, Kashiwaba Neurosurgical Hospital and Hokkaido Neurosurgical Memorial Hospital in Japan. The indications for surgery were >70% asymptomatic or symptomatic carotid artery stenosis. Patients were considered symptomatic if they had experienced an ischemic stroke or a TIA. Atherosclerotic plaques associated with symptoms were referred to as symptomatic or unstable plaques. The mean age of the 67 symptomatic patients was 73.2±6.9 years and that of the 15 asymptomatic patients was 71.3±7.6 years (NS). Patients whose excised CEA specimens were severely damaged were excluded from this analysis. This study, approved by the ETHICS COMMITTEE of each participating medical institution, complied with the Declaration of Helsinki on ethical principles for medical research involving human subjects, and all patients gave written informed consent to participate.

Histological Examinations

CEA was performed using standard surgical techniques with minimal handling of the specimens. The plaques were removed en bloc, fixed in 10% buffered formalin, transected transversely in 5-mm specimens, and embedded in paraffin. After hematoxylin-eosin and elastica-Masson (which stains elastin black and collagen and proteoglycans green) staining, 3–4 sections per specimen were examined. The sections with ulcerated plaques or thrombi, the most stenotic segments, or both, were retained for further analysis. The sections were examined by 2 independent observers, 1 of whom was an experienced histopathologist unaware of the clinical status and identity of the patient. The sections were probed with anti-CD31, anti-CD34 and PDGFRβ antibodies, which recognize endothelial cells, to confirm the presence of microvessels in the plaque. The factors we chose as potential predictors of unstable carotid plaque are listed in Appendix S1.

Definitions

Plaque rupture: an area of fibrous cap disruption, where the overlying thrombus is in continuity with the underlying NC.¹⁷

Plaque erosion: luminal thrombosis without communication between thrombus and NC.¹⁷

Thrombus: laminated platelets or fibrin, with or without interspersed red and white blood cells.

Calcified nodule: a plaque with luminal thrombi containing a calcific nodule protruding into the vessel lumen through a disrupted TFC.¹⁸

Intraplaque hemorrhage: microscopically visible blood and thrombus inside the plaque.

Intraplaque microvessels: presence of neovascularization in a carotid plaque.

Foamy macrophage within cap is a foam cell in the superficial intima.

Immature fibrillization: plaque stained light-green by elastica-Masson staining.

Necrotic core: acellular lipid pool within the intima, near the media where smooth muscle cells are scarce and proteoglycans and lipid deposition are abundant.¹⁷

Microcalcifications: calcifications 0.5–15 μm in diameter.^19,20

Fragmented or sheet calcification: aggregation of microcalcifications ≥15μm in diameter.^19,20

Nodular calcifications: breaks in calcified plates with fragments of calcium separated by fibrin.^19,21

Semiquantification of Pathological Observations

Because one of the characteristics of an unstable plaque is the presence of ≥25 macrophages per high-power (0.3-mm diameter) field (HPF),²² and because immunohistochemical staining has revealed that most of the inflammatory cells at the site of plaque rupture are macrophages,²³ we used ≥25 inflammatory cells/HPF as a threshold for plaque instability. Furthermore, because some studies have shown that (1) matrix metalloproteinase-12 (MMP-12) promotes atherosclerosis,^24,25 (2) the main source of MMP-12 is a foamy cap macrophage,^26,27 and (3) in patients undergoing CEA, the proportion of MMP-12 macrophages is approximately 10% of all macrophages,^26,28 we used ≥3 foamy or hemosiderin-laden macrophages/HPF as a threshold for plaque instability.

Cellular infiltration (i.e., ≥25 inflammatory cells/HPF) was scored semiquantitatively as absent (=0), limited to the fibrous cap or the shoulder of the cap (=1), or extending to the fibrous cap and the shoulder of the cap (=2) in >1 cross-sectional image of the lesion. Similarly, infiltration by ≥3 foamy or hemosiderin-laden macrophages/HPF was scored semiquantitatively as absent (=0), limited to the fibrous cap or the shoulder of the cap (=1), or abundant (=2) in the fibrous cap and the shoulder of the cap in ≥1 cross-sectional image of the lesion. The distribution of calcification was scored semiquantitatively as absent (0), 1–90° (1), 91–180° (2), or >180° (3) in ≥1 cross-sectional image of the lesion. Intraplaque hemorrhages were scored semiquantitatively as ≤1 (0), 2 (1), or ≥3 (2) cross-sections of the lesion.

Statistical Analysis

Continuous variables are reported as mean±standard deviation (SD) and categorical variables as counts and percentages. Between-group differences were analyzed using Pearson’s chi-square or Fisher’s exact test for categorical variables and Student’s t-test or Mann-Whitney U-test for continuous variables, as appropriate. The diagnostic value of the morphological characteristics of the plaque as predictors of symptomatic adverse cerebral events was examined by single-variable logistic regression analysis. Characteristics that emerged with P values (Wald statistics) <0.05 in the single-variable analysis were entered in the multiple variable regression analysis. Characteristics not defined for non-lipid plaques, such as TFC, were assigned a score of 0.

The score was derived from the multiple regression equation, to represent the probability of a correlation between a specific morphological characteristic of the plaque and a symptomatic lesion. The results are reported as P values, odds ratios (OR) and 95% confidence intervals (CI). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy and optimal cutoff values were calculated from receiver-operating characteristic curves. Values with the highest Youden-index (sensitivity+specificity−1) were identified as optimal cutoff values. A P-value <0.05 was considered to indicate statistical significance. The data were analyzed with the SPSS 22.0 statistical system software (IBM Corp., Armonk, NY, USA).

Results

Clinical and Pathological Characteristics of Symptomatic and Asymptomatic Patients Presenting With Unstable Plaques

The 82 patients enrolled in the study comprised 67 symptomatic and 15 asymptomatic patients. Plaques were resected from all patients and examined histopathologically. The clinical characteristics of the 67 symptomatic and 15 asymptomatic patients and the pathological characteristics of the lesions analyzed in each group are compared in Table 1. By single-variable analysis, several pathological characteristics were significantly associated with symptomatic manifestations: plaque rupture, minimum TFC overlying a NC, microcalcifications in the fibrous cap, intraplaque hemorrhage, intraplaque microvessels, hemosiderin-laden macrophages within the cap and an extensive NC. Figure 1 illustrates the main characteristics of plaque instability. Table 2 shows the prevalence of plaque rupture, erosion and a calcified nodule among patients presenting with stroke, vs. TIA, vs. no symptoms. The prevalence of plaque rupture was highest (73%) in patients presenting with stroke, significantly higher than in patients presenting with TIA (33%) or no symptoms (13%). In contrast, the prevalence of a calcified nodule was significantly higher in patients presenting with a TIA (25%) than in patients presenting with stroke (2%) or no symptom (0%).

Table 1. Clinical and Pathological Characteristics of Symptomatic and Asymptomatic Study Groups of Patients Undergoing Carotid Endarterectomy

	Symptomatic (n=67)	Asymptomatic (n=15)	OR (95% CI)	P value
Clinical characteristics
Age, years	73.2±6.9	71.3±7.6	1.88 (0.38–9.25)	0.667
Men	61 (91)	13 (87)	1.56 (0.28–8.64)	0.972
Diabetes mellitus	25 (37)	5 (33)	1.19 (0.37–3.88)	0.994
Hypertension	53 (79)	12 (80)	0.95 (0.23–3.82)	0.783
Dyslipidemia	57 (85)	11 (73)	2.07 (0.55–7.81)	0.476
Chronic kidney disease	17 (25)	8 (53)	0.30 (0.09–0.94)	0.069
Current smoker	22 (33)	5 (33)	0.98 (0.30–3.21)	0.790
History
TIA or cerebral infarction	15 (22)	3 (20)	1.15 (0.29–4.63)	0.886
Coronary artery disease	9 (13)	3 (20)	0.62 (0.15–2.64)	0.805
Peripheral artery disease	4 (6)	2 (13)	0.41 (0.07–2.50)	0.659
Prior drug therapy
Statin	9 (13)	5 (33)	0.31 (0.09–1.12)	0.141
Aspirin	6 (9)	4 (27)	0.27 (0.07–1.12)	0.145
Clopidogrel	3 (4)	2 (13)	0.30 (0.05–2.01)	0.485
Cilostazol	1 (1)	1 (7)	0.22 (0.01–3.60)	0.804
Days between onset of symptoms and operation	52±50	–		–
Admission laboratory data
Glycemia, mg/dL	136±51	128±55	2.06 (0.66–6.44)	0.343
Cholesterol, mg/dL
Low-density	118±32	101±25	4.33 (1.34–14.0)	0.025
High-density	52±12	54±17	0.18 (0.05–0.64)	0.015
LDL-C to HDL-C ratio	2.4±0.8	2.0±0.8	4.99 (1.48–16.8)	0.017
Triglyceride, mg/dL	156±76	191±113	0.31 (0.09–1.12)	0.141
Pathological characteristics
Plaque area, mm2	41.3±19.4	34.9±20.4	3.07 (0.97–9.71)	0.096
Cross-sectional area luminal narrowing, %	84.1±11.0	83.6±11.2	4.39 (0.54–36.1)	0.257
Plaque rupture	44 (66)	2 (13)	12.4 (2.58–59.9)	<0.001
Plaque erosion	9 (13)	4 (27)	0.43 (0.11–1.63)	0.380
Calcified nodule	4 (6)	0 (0)		0.759
Luminal thrombi	57 (85)	6 (40)	8.55 (2.49–29.3)	<0.001
Thinnest fibrous cap, μm	50.4±19.2	78.3±33.6	0.10 (0.03–0.36)	<0.001
Calcification	62 (93)	14 (93)	0.89 (0.10–8.19)	0.659
Extent of calcification	1.5±0.9	1.7±0.9	0.57 (0.18–1.79)	0.499
<15 μm microcalcification in the fibrous cap	53 (79)	7 (47)	4.33 (1.34–14.0)	0.025
Nodular calcification	26 (39)	2 (13)	4.12 (0.86–19.8)	0.114
Fragmented or sheet calcification	62 (93)	14 (93)	0.89 (0.10–8.19)	0.659
Maximum thickness of calcification, μm	679±552	799±746	0.35 (0.09–1.35)	0.201
Intraplaque hemorrhage	63 (94)	12 (80)	3.94 (1.04–19.5)	0.213
Intraplaque hemorrhage, sections	1.7±0.6	1.2±0.9	4.33 (1.34–14.0)	0.025
Intraplaque microvessels, /mm2	2.5±1.9	1.6±1.1	5.94 (1.24–28.4)	0.031
Inflammatory cells within cap	1.8±0.5	1.3±0.9	3.80 (1.11–13.0)	0.064
Foamy macrophages within cap	1.6±0.6	1.1±0.8	3.07 (0.97–9.71)	0.096
Hemosiderin-laden macrophages within cap	1.5±0.6	0.9±0.7	4.38 (1.13–16.9)	0.048
Immature fibrillization	66 (99)	13 (87)	10.2 (0.86–120)	0.148
Myxoid change in media	61 (91)	13 (87)	1.56 (0.28–8.64)	0.972
Necrotic core	63 (94)	13 (87)	2.42 (0.40–14.6)	0.659
>120° NC	58 (87)	9 (60)	4.30 (1.23–15.0)	0.042
Eccentric shape	58 (87)	13 (87)	0.99 (0.19–5.14)	0.683

Values are mean±SD or number (%) of observations. CI, confidence interval; OR, odds ratio; TIA, transient ischemic attack.

Figure 1.

Representative microscopic characteristics of unstable plaque. (A) Ruptured plaque with a luminal thrombus (black arrow; elastica-Masson). (B) Fibrous cap thickness measures 49.4 μm (yellow arrows). (C) Intraplaque microvessels stained by CD34 (arrowheads). (D) Another section shows microcalcifications in the fibrous cap (dashed arrows; H&E).

Table 2. Rates of Plaque Rupture, Erosion and a Calcified Nodule Among the Subgroups of Patients Undergoing Carotid Endarterectomy

	No. of plaques (%)			P value
	Major ipsilateral stroke (n=55)	TIA (n=12)	Asymptomatic (n=15)	Stroke vs.		TIA vs.
				TIA	asymptomatic	asymptomatic
Plaque rupture	40 (73)	4 (33)	2 (13)	0.023	<0.001	0.076
Plaque erosion	7 (13)	2 (17)	4 (27)	0.917	0.360	0.535
Calcified nodule	1 (2)	3 (25)	0	0.002	0.599	0.040

Values are mean±SD or numbers (%) of observations. TIA, transient ischemic attack.

By multiple-variable logistic regression analysis, the presence of plaque rupture, minimum TFC, the presence of microcalcifications in the fibrous cap and intraplaque microvessels were independent correlates of symptomatic plaques (Table 3).

Table 3. Pathological Lesion Characteristics Independently Associated With Symptomatic Ischemic Cerebrovascular Events

Variable	OR (95% CI)	P value
Presence of plaque rupture	9.75 (1.62–58.6)	0.013
Minimum thin fibrous cap (10 μm)	0.70 (0.51–0.96)	0.025
Presence of microcalcification in the fibrous cap	7.82 (1.35–45.4)	0.022
Intraplaque microvessels (/mm2)	1.91 (1.02–3.57)	0.043

CI, confidence interval; OR, odds ratio.

Diagnostic Performance of Predictors of Plaque Instability

We used receiver-operating characteristic statistics to examine the diagnostic performance of these indices of plaque instability (Figure 2; Table 4). Among all correlates of symptomatic cerebral ischemic event, minimum TFC had the highest diagnostic performance (area under the curve [AUC] 0.78, optimal cutoff value 70.2 μm, sensitivity 90%, specificity 60%, PPV 91%, NPV 56%, diagnostic accuracy 84%). However, combining the 4 independent correlates into a single score, using the equation derived from the multiple-variable logistic regression model (Logit(Score)=0.179+2.277* (insert 1 if plaque rupture present; otherwise 0)−0.355* (insert minimum TFC in multiples of 10 μm)+2.057* (insert 1 if microcalcification in the fibrous cap present; otherwise 0)+0.646* (insert intraplaque microvessels/mm²), increased the diagnostic performance to an AUC of 0.92 (95% CI 0.85–0.99; optimal cutoff value 0.814; sensitivity 90%; specificity 87%; PPV 97%; NPV 65%; diagnostic accuracy 89%). The application of this score to grade the instability of 2 separate lesions is illustrated in Figure 3.

Figure 2.

Pathological characteristics that identify symptomatic plaques individually as well as when combined in a score. Receiver-operating characteristic curves for (A) minimum fibrous cap thickness, (B) intraplaque microvessels to identify symptomatic plaque individually and (C) combined into a score. The score, which includes the presence of plaque rupture and microcalcifications in the fibrous cap, was calculated as described in the text.

Table 4. Outcome of Receiver-Operator Characteristics Analysis of Pathological Variables of Unstable Plaque

	AUC (95% CI)	Cutoff	Sensitivity	Specificity	Predictive value		Diagnostic accuracy
					Positive	Negative
Plaque rupture		Present	65.7	86.7	95.7	36.1	69.5
Minimum thin fibrous cap	0.78 (0.62–0.94)	70.2 μm	89.6	60.0	90.9	56.3	84.1
Fibrous cap microcalcifications		Present	79.1	53.3	88.3	36.4	74.4
Intraplaque microvessels	0.63 (0.49–0.77)	2.20/mm2	47.8	86.7	94.1	27.1	54.9
Score	0.92 (0.85–0.99)	0.814	89.6	86.7	96.8	65.0	89.0

AUC, area under the curve; CI, confidence interval.

Figure 3.

Examples of measurement of lesion instability. Representative images of unstable (A) and stable (B) carotid plaques quantified by the pathological score, with values indicated for the presence of plaque rupture, minimum thin fibrous cap (TFC), presence of microcalcifications in the cap and intraplaque microvessels. The score was calculated in these 2 examples and the absolute probability of symptomatic plaque is presented for each lesion.

Discussion

The main observations made in this study were (1) that although several pathological characteristics were associated with symptomatic plaques, plaque rupture, minimum TFC, microcalcifications in the fibrous cap and intraplaque microvessels were independent correlates, and (2) when combined into a single score, the performance of these 4 independent correlates in the diagnosis of pathologically unstable plaques in patients with carotid artery stenoses was very high. To the best of our knowledge, this study is the first to compare the pathological indices of unstable plaque by multiple-variable analysis, and then quantify the instability of carotid artery plaques in patients undergoing CEA.

Comparison With Previous Studies

Our pathological observations are concordant with those reported previously.^1,7,29,30 In this study, the proportion of symptomatic carotid plaques associated with plaque rupture was higher than that of asymptomatic plaques, and the prevalence of plaque rupture in patients presenting with stroke was higher than that of patients with presenting with a TIA or of asymptomatic patients. Previous studies found that symptomatic carotid artery disease is more often associated with plaque rupture than asymptomatic disease.^1,7,29,30 The prevalence of plaque rupture in stroke victims (67%) was significantly higher than in patients presenting with a TIA (23%) or in asymptomatic patients (13%).²⁹

Earlier studies have observed certain lesion morphologies more often in symptomatic than in asymptomatic patients undergoing CEA.^1,31,32 Histopathological studies have identified plaque rupture,^31,33 very TFC overlying lipid cores,^1,34 intimal foamy macrophages,^30,35 intraplaque hemorrhages^30,36 and intraplaque microvessels^37,38 as major characteristics of atherosclerotic plaque instability. Our own observations are concordant with those studies and add specific information, including the demonstration of a higher prevalence of plaque ruptures, thinner fibrous caps, the presence of microcalcifications in the fibrous cap, and a greater number of microvessels inside the plaques in symptomatic than in asymptomatic patients presenting with carotid artery stenoses.

Microcalcifications in the Fibrous Cap

This pathological study is the first to show that the presence of microcalcifications in the fibrous cap is an important sign of symptomatic carotid plaque. An increase in plaque structural stress increases the instability of atherosclerotic plaques.³⁹ Microcalcifications approximately 5–10 μm in diameter accumulate in the fibrous cap inside apoptotic smooth muscle cells or macrophages,⁴⁰ and superficial calcifications around or inside the fibrous cap increase the stress by nearly 50%.³⁹ When microcalcifications are clustered along the tensile axis of the cap in coronary arteries, they may increase the local structural stress over fivefold.⁴¹ In addition, microcalcifications typically 10 μm in diameter within a TFC promote plaque rupture by increasing the local stress and causing interfacial debonding.⁴⁰ Therefore, microcalcifications in the fibrous cap represent an additional factor of risk of carotid plaque instability.

Measuring Plaque Vulnerability

The individual and combined power of these plaque characteristics to measure plaque instability is incompletely understood. In this study, we combined these previously described individual characteristics of plaque instability into a single score. From our comprehensive pathological study and multiple-variable analysis, we identified the factors correlating with plaque instability. Among all the pathological indices, plaque rupture, minimum TFC, presence of microcalcifications and intraplaque microvessels were the only independent correlates of symptomatic plaque in this sample of patients with carotid artery stenosis. Among all the pathological factors we examined, minimum TFC was the most accurate to quantify plaque instability. Furthermore, when combined into a score, these risk factors were highly accurate to quantify plaque instability and identify unstable plaques in patients with carotid artery stenoses. Although plaque rupture has been described as one of the major features of unstable plaque in histological studies,^1,4,30 plaque rupture is not the only characteristic of an unstable atherosclerotic lesion. A representative example of unstable plaque without plaque rupture is shown in Figure S1.

Insignificant Variables by Multiple-Variable Analysis

Intraplaque hemorrhages, hemosiderin-laden macrophages and extensive NC, although related to unstable plaques by single-variable analysis, were no longer statistically significant by multiple-variable analysis. Although plaques removed ≤60 days after the most recent event are more unstable after a stroke than after a TIA, the instability persists after a TIA, and plaques removed >180 days after a recent event are less unstable after a stroke than after a TIA.³⁰ Because >80% of symptomatic patients presented with major ipsilateral stroke in this study, and the average timing of CEA was 52±50 days after the onset of symptoms, intraplaque hemorrhages, inflammatory cells and extensive NC might no longer be significant factors because of the long interval between the onset of symptoms and CEA. In contrast, because the statistical significance between plaque instability and intraplaque microvessels is not related to the time since onset of stroke,³⁰ it might remain an independent predictor of unstable plaque by multiple-variable analysis. Furthermore, intraplaque hemorrhages have been correlated with plaque rupture in symptomatic carotid artery stenosis.¹ It is noteworthy that plaque rupture and intraplaque hemorrhages were correlated in this study (r=0.362; r²=0.131; P<0.001). Therefore, it might be a confounding factor in the multiple-variable analysis.

Higher Prevalence of Calcified Nodules in Cases of TIA Than of Stroke

Calcified nodules might be associated with more stable atherosclerotic lesions than plaque rupture in symptomatic patients. Several matrix metalloproteinases produced by activated macrophages digest fibrillar collagen, thus reducing the plaque’s mechanical stability.⁴² Our comparisons of plaques with rupture and with calcified nodules showed significantly less severe infiltration of inflammatory cells (1.9±0.4 vs. 1.3±0.9, P=0.015) and foamy macrophages (1.7±0.5 vs. 1.0±0.8, P=0.037) in those with a calcified nodule (Table S1). Therefore, calcified nodules might be more frequently observed in patients with TIA, compared with those with stroke. As for coronary artery disease, Jia et al showed in an optical coherence tomography (OCT) analysis that the prevalence of a calcified nodule was higher in patients with non-ST-segment elevation myocardial infarction than in those with ST-segment elevation myocardial infarction (15% vs. 0%, P=0.004).⁴³

Clinical Perspectives

First, applying the indices of plaque instability derived in this study using techniques such as 3-dimensional or contrast-enhanced ultrasonography,^11,44 computed tomography (CT),⁴⁵ magnetic resonance imaging (MRI),^44,46 and positron-emission tomography (PET),⁴⁷ which can be used to measure these indices, may supplement conventional plaque imaging and improve our prediction of adverse CVE. High-resolution MRI is the best means of imaging carotid plaques and can effectively visualize plaque rupture, intraplaque hemorrhages and TFC; 7-tesla MRI might enable further detailed visualization of the structure of carotid plaques.⁴⁸ Contrast-enhanced ultrasound allows quantification of intraplaque neovascularization as a feature of instability of carotid plaque, and has been closely correlated with histopathological observations.⁴⁹ The 1-μm resolution of OCT is capable of revealing microcalcifications as the accumulation of small, punctate high-density signals within the fibrous cap.¹² These microcalcifications in the atherosclerotic plaque can also be identified with¹⁸ sodium fluoride PET or¹⁸ sodium fluoride PET/CT.⁵⁰ However, further technological developments are needed, including of spatial resolution, to detect unstable plaques more precisely. Second, we can verify the consistency between a preoperative diagnosis made with these imaging techniques and the plaque instability quantified by our novel pathological score. In our study, the plaque instability diagnosed by preoperative examinations including ultrasonography, MRI and CT was consistent with the pathological analysis of plaque instability in 72 of 82 patients (88%). Third, the plaque instability score might be a predictor of CVE. During the 6-month follow-up of, CVE occurred in 2 of 55 patients presenting with major ipsilateral stroke. The mean instability score was 97.8±2.3% in patients with CVE compared with 90.2±17.1% in those without CVE (P=0.329, Table S2). Fourth, carotid artery plaques with certain morphological features might be better targets for carotid artery stenting (CAS). Previous studies have shown that some morphological plaque features are associated with cerebrovascular complications pertaining to CAS. Tsutsumi et al showed that severe calcification of the carotid artery might be a cause of incomplete stent expansion despite aggressive dilatation.⁵¹ Bicknell and Cheshire reported that lipid-rich plaque was an independent predictor of distal embolization during the procedure.⁵² Furthermore, Kolodgie et al described extensive inflammation as an important risk factor of restenosis after CAS.²¹ In our study, age and the thickness of calcification were positively correlated (Figure S2). Infiltration of inflammatory cells, as well as foamy and hemosiderin-laden macrophages, was less observed in patients with NC <120° than in those with NC ≥120° (Table S3). These observations suggest that non-elderly patients without severe calcification or extensive NC in plaque would be likely to preferentially benefit from CAS. Fifth, several studies have shown that the mechanisms of carotid plaque instability are similar to those of plaque found in the coronary circulation,^29,30 although, unlike the myocardial circulation, the carotid vascular bed is exposed to a high blood flow. Therefore, this study should provide useful information, including the identification of unstable plaques, about patients presenting with coronary artery disease.

Study Limitations

The sample size of this retrospective, observational study, conducted at 3 medical centers, was small. Our results need to be confirmed by a study including a larger number of patients. Second, in our population, the median time between the last adverse clinical event and CEA was 52±50 days, whereas the current professional practice guidelines recommend that patients presenting with symptomatic carotid stenosis undergo CEA within 14 days of the last event. Therefore, in further histopathological analyses, factors such as infiltration by inflammatory cells could turn out to be strong predictors of unstable plaques in patients undergoing CEA. Third, instead of being functional, this study was mainly a quantitative, morphological analysis of excised specimens of carotid plaques, which did not include measurements of enzymatic concentrations derived from foamy macrophages, such as that of matrix metalloproteinase.³⁵ Fourth, although observations that are not derived from atherosclerotic plaques (e.g., wall shear stress) have also been found to predict plaque instability,⁵³ this study was limited to pathological indices to quantify plaque instability.

Conclusions

This pathological analysis of carotid artery plaques suggested that our diagnostic scoring may facilitate understanding of plaque instability. Pathological quantification by this scoring simplified the assessment of carotid artery plaques, instead of requiring the wide variety of pathological characteristics reflecting plaque instability.

Acknowledgments

We thank Ms. Naomi Kobayashi for her assistance in the data collection, Dr. Souichirou Seno for his useful information about neurosurgery and Ms. Tomoko Takenami and Ms. Masana Urushido for their contributions in the immunohistological staining.

Acknowledgment of Grant Support

None.

Disclosures

The authors have no potential conflict of interest to disclose.

Supplementary Files

Supplementary File 1

Appendix S1. Potential Characteristics of Unstable Plaque

Figure S1. Representative example of unstable plaque without plaque rupture.

Figure S2. Positive correlation between age and maximum thickness of calcification (r=0.378, r²=0.143, P<0.001) in patients undergoing carotid endarterectomy.

Table S1. Comparison of plaque rupture and calcified nodule groups of patients undergoing carotid endarterectomy

Table S2. Comparison of CVE and no CVE groups of patients undergoing carotid endarterectomy

Table S3. Comparisons of NC ≤120° and NC >120° groups of patients undergoing carotid endarterectomy

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-17-0204

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