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The Role of Carotid Plaque Vulnerability and Inflammation in the Pathogenesis of Acute Ischemic Stroke
The Role of Carotid Plaque Vulnerability and Inflammation in the Pathogenesis of Acute Ischemic Stroke SHIFANG DING, MD, PHD; MEI ZHANG, MD, PHD; YUXIA ZHAO, MD, PHD; WENQIANG CHEN, MD, PHD; GUIHUA YAO, MD, PHD; CHENG ZHANG, MD, PHD; PENGFEI ZHANG, MD, PHD; YUN ZHANG, MD, PHD
ABSTRACT: Background: Increasing evidences show that disruption of carotid plaque followed by arterioarterial thromboembolism is an important mechanism in the generation of ischemic stroke. Inflammatory mechanisms play a key role in transforming structurally vulnerable plaques into functionally unstable ones. The purpose of the present study is to evaluate the roles of carotid plaque vulnerability and inflammation in the pathogenesis of acute ischemic stroke. Methods: Fiftytwo patients with acute ischemic stroke affecting the anterior circulation (stroke group) and 44 with asymptomatic carotid stenosis (asymptomatic group) were investigated. Duplex ultrasonography was used to evaluate the characteristics of carotid plaque and grading the degree of carotid stenosis. Plaque echogenicity was assessed as echolucent, predominantly echolucent, predominantly echogenic, or echogenic. Plaque surface was classified as smooth, irregular, or ulcerated. All subjects had duplex-determined 50% to 99% carotid stenosis. Serum levels of matrix metalloproteinase-9
(MMP-9), tissue inhibitors of metalloproteinases (TIMP1), soluble CD40 ligand (sCD40L) and high-sensitivity C-reactive protein (hsCRP) were measured. Results: Plaques in the stroke group were echolucent or predominantly echolucent, whereas those of the asymptomatic group were predominantly echogenic or echogenic plaques (P ⬍ 0.05). Irregular and ulcerated plaques were frequently found in stroke patients, while smooth plaques were frequently detected in asymptomatic patients (P ⬍ 0.05). Serum levels of MMP-9, sCD40L, hsCRP were higher in stroke than in asymptomatic patients. By contrast, serum TIMP-1 levels were significantly higher in the asymptomatic than in the stroke group. Conclusions: The results suggest that inflammation plays a crucial role in carotid plaque vulnerability and, together with carotid plaque morphology, in the pathogenesis of acute ischemic stroke. KEY INDEXING TERMS: Inflammation; Carotid arteries; Plaque; Stroke; Ischemic; Ultrasonography. [Am J Med Sci 2008;336(1):27–31.]
T
the severity of carotid stenosis might not be sufficient to detect patients at high risk to develop ischemic stroke. Instead, the identification of factors other than degree of stenosis may improve risk stratification and help to decrease the likelihood of stroke in the carotid territory.5 The term “vulnerable plaque” has been used to describe rupture-prone plaques that have a large lipid pool (⬎40% of the plaque’s total volume), a thin cap ⬍100 m, and extensive macrophage accumulation.6 Analogous to acute myocardial infarction, recent studies show that the sequence of vulnerable plaque rupture, surface thrombus formation, and distal embolism is an important pathophysiologic of large artery ischemic stroke.7 Inflammatory mechanisms play a key role in the transformation of structurally vulnerable plaques into functionally unstable ones. Thus, to evaluate plaque vulnerability, an ideal approach would provide information regarding both the anatomic characteristics (morphology) of the plaque as
he benefit of carotid endarterectomy in patients with severe carotid stenosis has been demonstrated by 3 large-scale, multicenter, randomized trials.1–3 However, many patients, even if selected appropriately, might not receive the full benefit of carotid endarterectomy.4 These results suggest that From the Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, Jinan, Shandong, People’s Republic of China. Submitted February 28, 2007; accepted in revised form September 17, 2007. This study was supported by the National Natural Science Foundation of China (30470702, 30570747), and the Key Clinical Project of the Chinese Ministry of Health (No. 20012943). Correspondence: Yun Zhang, MD, PhD, The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, No. 107 Wen Hua Xi Road, Jinan, Shandong 250012, People’s Republic of China (E-mail: zhangyun@ sdu.edu.cn). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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Carotid Plaque Vulnerability and Ischemic Stroke
well as its functional properties (activity).6 In our study, carotid plaque morphology was examined using high-resolution B-mode ultrasonography, and serum levels of inflammatory biomarkers were measured. Based on this information, the role of inflammation and destabilization of the carotid plaque in the pathogenesis of acute ischemic stroke was determined. Subjects and Methods Study Population The subjects for this cross-sectional study were enrolled from patients in the Department of Neurology at Qilu hospital between May 2003 and May 2004. The stroke group consisted of 52 patients with first-ever acute hemispheric ischemic stroke of the anterior circulation who underwent MR imaging within 24 hours of the onset of symptoms.8 In addition, 44 asymptomatic patients (asymptomatic group) were also studied who never had symptoms such as amaurosis fugax, transient ischemic attacks, or stroke. At the baseline examination, cardiovascular risk factors including hypertension (blood pressure ⱖ140/90 mm Hg or current use of antihypertensive agents), smoking status (ⱖ10 cigarettes per day for ⬎1 year), and diabetes (fasting blood glucose ⱖ7.0 mmol/L or use of medication for diabetes) were documented and collected through interviews. The following patients were excluded from the study: (1) patients with cerebral hemorrhage, lacunar infarcts, silent infarcts, or posterior circulation infarcts; (2) patients with disorders commonly associated with possible or probable sources of cardioembolism, including atrial fibrillation, cardiac valve abnormalities, recent myocardial infarction, or heart failure; (3) in case of bilateral stenoses, patients with the highest degree of “culprit” carotid stenosis ⬍50%; (4) patients with concurrent diseases or conditions interfering with the expression of inflammatory mediators, such as trauma, surgery or ischemic events during the previous 3 months, severe liver disease, renal failure, hematologic, or malignant diseases, chronic inflammatory diseases, autoimmune diseases, as well as patients with fever or infectious conditions at study entry. The Medical Ethics Committee of Qilu Hospital approved the study, and all patients had to provide written informed consent.
Carotid Ultrasound Examination In symptomatic patients, carotid ultrasound examinations were carried out within 24 hours after onset of symptoms. Ultrasound images were achieved with a 7.5-MHz linear-array transducer (Phillips SONOS 5500). The subject lay in the supine position, with his or her head held in the midline position or slightly tilted to either side. Three different longitudinal views (anterior oblique, lateral, and posterior oblique) and transverse views of both carotid systems were scanned by B-mode and color Doppler methods. Grading of carotid stenosis was determined by routine Doppler criteria as ⬍50%, 50% to 79%, 80% to 99%, or occlusion. Doppler systolic velocity ⬎120 cm/s were used to estimate ⱖ50% stenosis, and a diastolic velocity ⬎135 cm/s indicated ⱖ80% stenosis.9 An atherosclerotic plaque was defined as a focal protrusion 50% greater than the surrounding area and localized thickening ⬎1.2 mm along the extracranial carotid tree. Plaque morphology in terms of echogenicity was graded from 1 to 4 as echolucent, predominantly echolucent, predominantly echogenic, or echogenic, respectively.10 The vessel lumen was used as the reference structure for defining echolucency, and the bright echo zone produced by the media-adventitia interface in the far wall was used as the reference structure for defining echogenicity. Plaque surface structure was classified into 3 categories: smooth, irregular (height variations between 0.4 mm and 2 mm), or ulcerated (ulceration must meet the following criteria: discrete depression ⬎2 mm in width extending into the media, as well as
28
Figure 1. Echolucent plaque with ulceration.
exhibiting an area of reversed flow within the recess or a zone of low flow signal at the level of the recess). In subjects with multiple plaques, only the thickest plaque was selected for analysis. All ultrasound examinations were performed by the same experienced ultrasonographer who was unaware of history and physical findings of the patient, and images of the carotid artery plaques were recorded on magneto-optical discs for further analyses. Representative ultrasound images of atherosclerotic carotid plaques are shown in Figures 1, 2 and 3. To determine the reproducibility of ultrasonic characterization of plaque morphology, a second independent observer reviewed each plaque, and each was blinded to the results of the other. Intra- and interobserver reproducibility of plaque morphology characteristics were greater than 0.80 in our laboratory.
Measurement of Serum Inflammatory Markers Venous blood was collected between 6:00 and 7:00 AM after an overnight fast. Peripheral venous blood was drawn into pyrogenfree blood-collection tubes without any additives and allowed to clot at room temperature for 30 minutes before centrifugation (3500 rpm and 4°C for 15 minutes). Serum was stored at ⫺80°C until analyzed, and the samples were thawed only once. Highsensitivity C-reactive protein (hsCRP) was determined by an ultrasensitive turbidity test based on latex beads (Orion Diagnostica). Serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinases (TIMP-1), and soluble CD40 ligand (sCD40L) were determined with an enzyme-linked immunosorbent assay (MMP-9: R&D Systems; TIMP-1: R&D Systems; sCD40L: Bender MedSystems). Serum levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol were determined using standard automated procedures with commercial kits (Zhejiang dongou technology company, Wenzhou, China). All measurements were done according to the manufacturer’s instructions by laboratory staff unaware of the clinical data.
Figure 2. Predominantly echolucent plaque with ulceration.
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Table 2. Morphological Characteristics of Carotid Plaques
Characteristics
Figure 3. Predominantly echogenic plaque with irregular surface.
Carotid stenosis 50%–79% 80%–99% Echogenicity Echolucent Predominantly echolucent Predominantly echogenic Echogenic Surface irregularity Smooth Irregularity Ulceration
Stroke Group (n ⫽ 52)
Asymptomatic Group (n ⫽ 44)
34 18
30 14
29 10
11 9
6
15
7
9
14 22 16
26 15 3
P 0.772 0.009
0.001
Statistical Analysis All statistical tests have been performed with the SPSS program, version 10.0. Probability values of P ⬍ 0.05 were considered statistically significant. Continuous variables were compared using the Student t test. Categorical variables were compared with the Pearson 2 test.
Results Baseline Clinical Characteristics Table 1 summarizes the age, gender, conventional cardiovascular risk factors, and lipid profiles of participants. Patient characteristics were comparable between the stroke group and the asymptomatic group (P ⬎ 0.05). Ultrasound Analysis of Carotid Plaque Morphology Table 2 shows the results of carotid plaque morphology and carotid stenoses in the stroke and the asymptomatic groups. The distribution of carotid stenosis was similar between stroke patients and asymptomatic patients (P ⬎ 0.05). However, echolucent and predominantly echolucent plaques were more frequently found in stroke patients, whereas
echogenic and predominantly echogenic plaques were seen in asymptomatic patients (P ⬍ 0.05). Smooth plaques were more often observed in asymptomatic patients, whereas irregular and ulcerated plaques occurred more frequently in stroke patients (P ⬍ 0.05). Serum Levels of Inflammatory Markers Table 3 shows the serum levels of inflammatory markers in 2 groups. The serum levels of hsCR, sCD40L, MMP-9 levels were higher in the stroke group than the asymptomatic group (P ⬍ 0.05), whereas serum TIMP-1 levels were higher in the asymptomatic group than in the stroke group (P ⬍ 0.05). Discussion Carotid plaque echogenicity, as assessed by the noninvasive techniques of duplex ultrasonography, is consistent with its histologic characteristics.11 Echolucent plaques are commonly characterized by a large necrotic/lipid core with a thin fibrous cap, whereas echogenic plaques have a higher content of
Table 1. Baseline Characteristics of the Study Participants
Characteristics
Stroke Group (n ⫽ 52)
Asymptomatic Group (n ⫽ 44)
Age (yrs) Male/Females Smokers (%) Diabetes mellitus (%) Hypertension (%) Family history (%) Total cholesterol (mmol/L) Triglycerides (mmol/L) LDL cholesterol (mmol/L) HDL cholesterol (mmol/L)
63.92 ⫾ 9.72 34/18 19 (36.5) 15 (28.8) 30 (57.7) 8 (15.4) 4.91 ⫾ 1.14 1.64 ⫾ 0.95 3.19 ⫾ 0.79 1.07 ⫾ 0.29
61.43 ⫾ 4.39 32/12 18 (40.9) 16 (36.4) 22 (50.0) 6 (13.6) 4.70 ⫾ 1.34 1.84 ⫾ 1.42 3.06 ⫾ 0.90 1.02 ⫾ 0.23
Continuous normally distributed data are shown as mean ⫾ SD. LDL, low-density lipoprotein; HDL, high-density lipoprotein.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Table 3. Serum Levels of Inflammatory Markers
Characteristics
Stroke Group (n ⫽ 52)
Asymptomatic Group (n ⫽ 44)
hsCRP (mg/L) MMP-9 (ng/mL) TIMP-1 (ng/mL) sCD40L (pg/mL)
5.23 ⫾ 2.18* 260.88 ⫾ 92.80* 58.22 ⫾ 14.95* 10.49 ⫾ 3.56*
1.80 ⫾ 0.34 144.67 ⫾ 34.40 73.48 ⫾ 21.22 7.18 ⫾ 2.77
Continuous normally distributed data are shown as mean ⫾ SD. * Compared with asymptomatic group. hs-CRP, high sensitivity C-reactive protein; MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metalloproteinases; sCD40, soluble CD40 ligand.
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Carotid Plaque Vulnerability and Ischemic Stroke
fibrous tissue and calcification. Echolucent plaques are structurally vulnerable, and several studies have shown that they are associated with an increased risk for ischemic stroke and other cerebrovascular events.12 Gray-Weale et al9 reported that cerebrovascular events were mainly present in patients with either echolucent plaques or echolucent with small echogenic plaques, whereas echogenic plaques with small echolucent areas and plain echogenic plaques were mainly asymptomatic. Grønholdt et al10 reported that echolucent plaques causing ⱖ50% stenosis of the vessel diameter were associated with a higher risk of future stroke in symptomatic but not in asymptomatic individuals. Mathiesen et al13 reported that echolucent atherosclerotic plaques increased the risk of ischemic cerebrovascular events. Recently, Yamashiro et al14 reported that echolucent plaques were more frequently observed in atherothrombotic stroke. In the current study, echolucent and predominantly echolucent carotid plaques were more frequently found in the stroke than in the asymptomatic group of patients. Regarding the significance of the carotid plaque surface, ulceration causes exposure of the thrombogenic lipid core, leading to subsequent intraluminal thrombus formation and, eventually, emboli.15,16 One prospective study17 observed that presence of an irregular plaque was an independent predictor of ischemic stroke risk (adjusted HR, 3.1; 95% CI, 1.1– 8.5). In our study, irregular and ulcerated plaques were detected with greater frequency in the stroke group than in the asymptomatic group. However, other sonographic studies have shown that it was the deep ulceration of plaques and not their surface irregularity, is associated with the presence of symptoms.18 These discordant findings may be related to different definitions of plaque morphology. Disruption of vulnerable carotid plaques, with subsequent arterio-arterial thromboembolism, is an important mechanism of ischemic stroke. However, plaque vulnerability has been shown to be related to the increased local number of inflammatory cells within the plaque, and in particular, to extensive macrophage accumulation.19 MMPs are synthesized and released mainly by activated macrophages, and are capable of degrading all extracellular matrix components. In particular, 92-kDa gelatinase (MMP-9) has been critically associated with carotid plaque vulnerability.20 The activity of these enzymes is tightly controlled by endogenous inhibitors known as TIMPs.21 TIMP-1 is a 28.5kDa glycoprotein that inhibits the activity of MMPs, with a particular affinity for MMP-1, -2, and -9. The degradation of the plaque’s fibrous cap is dependent on the balance between activated MMPs and TIMPs. A recent study by Loftus et al22 indicated a localized increase in the concentration 30
of MMP-9 in the most unstable carotid plaques from symptomatic patients compared with plaques from asymptomatic patients. However, there were no significant differences in the level of TIMP-1 between the 2 groups. Our results were consistent with the findings of Sapienza et al,23 who reported that plasma levels of MMPs were higher in patients with unstable plaques than in patients with stable plaques (P ⬍ 0.001). By contrast, plasma levels of TIMPs were lower in patients with unstable plaques than in patients with stable plaques (P ⬍ 0.001). These results demonstrate that an imbalance between the activities of MMP-9 and TIMP-1 may be responsible for the different clinical manifestations in the stroke group compared with the asymptomatic group. Atherosclerotic plaque rupture itself does not always lead to symptoms, but rather vulnerable blood (prone to thrombosis) with subsequent large thrombus formation plays a key role in the various clinical presentations.24,25 Activation of platelets is a crucial mediator of arterial thrombosis and increased platelet activity has been demonstrated by measuring serum levels of platelet-released products.26 CD40 ligand (CD40L) was found to be expressed on the surface of platelets. It can also be cleaved from cell membranes to form a soluble fragment, sCD40L, which results in the activation of MMPs and procoagulant activity.27 Activated platelets are the major source of sCD40L, and elevated levels of the fragment were reportedly associated with acute coronary syndromes and ischemic stroke.28 In the present study, stroke patients had significantly higher levels of sCD40L compared with asymptomatic patients, suggesting that the increased serum level of sCD40L played a role in platelet activation and atherothrombosis in ischemic stroke. Atherosclerosis is currently recognized as an inflammatory disease, and inflammation has a crucial role in plaque rupture.7,24 C-reactive protein is not only a nonspecific biomarker of systemic inflammation, but also is a contributor to atheroma development, progression, and instability. Serum hsCRP levels provide a means to identify patients at risk for plaque rupture as well as those with a poor prognosis after plaque rupture. Our finding that hsCRP was higher in ischemic stroke than in the asymptomatic patients suggests that systemic inflammation may be responsible for carotid atherosclerosis.29 Study Limitations Our study and the interpretation of our results have several limitations. First, we did not obtain endarterectomy specimens, and serum inflammatory markers were measured only once. Second, other vascular studies, including transcranial Doppler examination, or magnetic resonance angiography, or computed tomography angiography were not carried out to provide further information on the July 2008 Volume 336 Number 1
Ding et al
nature of intracranial large-artery disease. Third, plaque characterization by Doppler ultrasound is usually a subjective process that is observer dependent.30 Finally, this study was cross-sectionally designed and the sample size was small, and further investigation is needed to confirm the clinical significance of the findings.
14. 15.
16.
Conclusions We conclude that structurally vulnerable carotid plaque is closely related to the pathogenesis of ischemic stroke in patients with carotid stenosis, and that inflammation plays a key role in carotid plaque rupture and subsequent atherothrombosis.
17.
References
19.
1. North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med 1991;325:445–53. 2. European Carotid Trial Collaborative Group. MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (70 –99%) or mild (0 –29%) carotid stenosis. Lancet 1991;337:1235– 43. 3. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study. Endarterectomy for asymptomatic carotid artery stenosis. JAMA 1995;273:1421– 8. 4. Chassin MR. Appropriate use of carotid endarterectomy. N Engl J Med 1998;339:1468 –71. 5. Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke 2000;31:774 – 81. 6. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I. Circulation 2003;108:1664 –72. 7. Hollander M, Bots ML, del Sol AI, et al. Carotid plaques increase the risk of stroke and subtypes of cerebral infarction in asymptomatic elderly: the Rotterdam study. Circulation 2002;105:2872–7. 8. Bamford J, Sandercock P, Dennis M, et al. Classification and natural history of clinically identifiable subtypes of cerebral infarction. Lancet 1991;337:1521– 6. 9. Grønholdt ML, Nordestgaard BG, Schroeder TV, et al. Ultrasonic echolucent carotid plaques predict future stroke. Circulation 2001;104:68 –73. 10. Gray-Weale AC, Graham JC, Burnett JR, et al. Carotid artery atheroma: comparison of preoperative B-mode ultrasound appearance with carotid endarterectomy specimen pathology. J Cardiovasc Surg 1988;29:676 – 81. 11. Tegos TJ, Sohail M, Sabetai MM, et al. Echomorphologic and histopathologic characteristics of unstable carotid plaques. AJNR Am J Neuroradiol 2000;21:1937– 44. 12. Gorgan JK, Shaalan WE, Cheng H, et al. B-mode ultrasonographic characterization of carotid atherosclerotic plaques in symptomatic and asymptomatic patients. J Vasc Surg 2005;42: 435– 41. 13. Mathiesen EB, Bønaa KH, Joakimsen O. Echolucent plaques are associated with high risk of ischemic cerebrovas-
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cular events in carotid stenosis: the Tromsø study. Circulation 2001;103:2171–5. Yamashiro K, Watanabe T, Tanaka R, et al. Clustering of risk factors increases the incidence of echolucent carotid plaque in stroke patients. Cerebrovasc Dis 2006;22:432– 8. Sitzer M, Mu ¨ ller W, Siebler M, et al. Plaque ulceration and lumen thrombus are the main sources of cerebral microemboli in high-grade internal carotid artery stenosis. Stroke 1995;26:1231–3. Fisher M, Paganini-Hill A, Martin A, et al. Carotid plaque pathology: thrombosis, ulceration, and stroke pathogenesis. Stroke 2005;36:253–7. Prabhakaran S, Rundek T, Ramas R, et al. Carotid plaque surface irregularity predicts ischemic stroke: The Northern Manhattan Study. Stroke 2006;37:2696 –701. Johnson JM, Ansel AL, Morgan S, et al. Ultrasonographic screening for evaluation and follow-up of carotid artery ulceration: a new basis for assessing risk. Am J Surg 1982;144: 614 – 8. Jander S, Sitzer M, Schumann R, et al. Inflammation in high-grade carotid stenosis: a possible role for macrophages and T cells in plaque destabilization. Stroke 1998; 29:1625–30. Sluijter JP, Pulskens WP, Schoneveld AH, et al. Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms. Stroke 2006;37: 235–9. Loftus IM, Naylor AR, Bell PR, et al. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg 2002;89:680 –94. Loftus IM, Naylor R, Goodall S, et al. Increased matrix metalloproteinase-9 activity in unstable carotid plaques: a potential role in acute plaque disruption. Stroke 2000;31:40 –7. Sapienza P, di Marzo L, Borrelli V, et al. Metalloproteinases and their inhibitors are markers of plaque instability. Surgery 2005;137:355– 63. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II. Circulation 2003;108:1772– 8. Fujii K, Kobayashi Y, Mintz GS, et al. Intravascular ultrasound assessment of ulcerated ruptured plaques: a comparison of culprit and nonculprit lesions of patients with acute coronary syndromes and lesions in patients without acute coronary syndromes. Circulation 2003;108:2473– 8. Cherian P, Hankey GJ, Eikelboom JW, et al. Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke 2003;34:2132–7. Inwald DP, McDowall A, Peters MJ, et al. CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res 2003;92:1041– 8. Garlichs CD, Kozina S, Fateh-Moghadam S, et al. Upregulation of CD40-CD40 Ligand (CD154) in patients with acute cerebral ischemia. Stroke 2003;34:1412– 8. Napoli MD, Papa F, Bocola V. Prognostic influence of increased C-reactive protein and fibrinogen levels in ischemic stroke. Stroke 2001;32:133– 8. Sabetai MM, Tegos TJ, Nicolaides AN, et al. Reproducibility of computer-quantified carotid plaque echogenicity: can we overcome the subjectivity? Stroke 2000;31:2189 –96.
31
ABSTRACT: Background: Increasing evidences show that disruption of carotid plaque followed by arterioarterial thromboembolism is an important mechanism in the generation of ischemic stroke. Inflammatory mechanisms play a key role in transforming structurally vulnerable plaques into functionally unstable ones. The purpose of the present study is to evaluate the roles of carotid plaque vulnerability and inflammation in the pathogenesis of acute ischemic stroke. Methods: Fiftytwo patients with acute ischemic stroke affecting the anterior circulation (stroke group) and 44 with asymptomatic carotid stenosis (asymptomatic group) were investigated. Duplex ultrasonography was used to evaluate the characteristics of carotid plaque and grading the degree of carotid stenosis. Plaque echogenicity was assessed as echolucent, predominantly echolucent, predominantly echogenic, or echogenic. Plaque surface was classified as smooth, irregular, or ulcerated. All subjects had duplex-determined 50% to 99% carotid stenosis. Serum levels of matrix metalloproteinase-9
(MMP-9), tissue inhibitors of metalloproteinases (TIMP1), soluble CD40 ligand (sCD40L) and high-sensitivity C-reactive protein (hsCRP) were measured. Results: Plaques in the stroke group were echolucent or predominantly echolucent, whereas those of the asymptomatic group were predominantly echogenic or echogenic plaques (P ⬍ 0.05). Irregular and ulcerated plaques were frequently found in stroke patients, while smooth plaques were frequently detected in asymptomatic patients (P ⬍ 0.05). Serum levels of MMP-9, sCD40L, hsCRP were higher in stroke than in asymptomatic patients. By contrast, serum TIMP-1 levels were significantly higher in the asymptomatic than in the stroke group. Conclusions: The results suggest that inflammation plays a crucial role in carotid plaque vulnerability and, together with carotid plaque morphology, in the pathogenesis of acute ischemic stroke. KEY INDEXING TERMS: Inflammation; Carotid arteries; Plaque; Stroke; Ischemic; Ultrasonography. [Am J Med Sci 2008;336(1):27–31.]
T
the severity of carotid stenosis might not be sufficient to detect patients at high risk to develop ischemic stroke. Instead, the identification of factors other than degree of stenosis may improve risk stratification and help to decrease the likelihood of stroke in the carotid territory.5 The term “vulnerable plaque” has been used to describe rupture-prone plaques that have a large lipid pool (⬎40% of the plaque’s total volume), a thin cap ⬍100 m, and extensive macrophage accumulation.6 Analogous to acute myocardial infarction, recent studies show that the sequence of vulnerable plaque rupture, surface thrombus formation, and distal embolism is an important pathophysiologic of large artery ischemic stroke.7 Inflammatory mechanisms play a key role in the transformation of structurally vulnerable plaques into functionally unstable ones. Thus, to evaluate plaque vulnerability, an ideal approach would provide information regarding both the anatomic characteristics (morphology) of the plaque as
he benefit of carotid endarterectomy in patients with severe carotid stenosis has been demonstrated by 3 large-scale, multicenter, randomized trials.1–3 However, many patients, even if selected appropriately, might not receive the full benefit of carotid endarterectomy.4 These results suggest that From the Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, Jinan, Shandong, People’s Republic of China. Submitted February 28, 2007; accepted in revised form September 17, 2007. This study was supported by the National Natural Science Foundation of China (30470702, 30570747), and the Key Clinical Project of the Chinese Ministry of Health (No. 20012943). Correspondence: Yun Zhang, MD, PhD, The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, No. 107 Wen Hua Xi Road, Jinan, Shandong 250012, People’s Republic of China (E-mail: zhangyun@ sdu.edu.cn). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
27
Carotid Plaque Vulnerability and Ischemic Stroke
well as its functional properties (activity).6 In our study, carotid plaque morphology was examined using high-resolution B-mode ultrasonography, and serum levels of inflammatory biomarkers were measured. Based on this information, the role of inflammation and destabilization of the carotid plaque in the pathogenesis of acute ischemic stroke was determined. Subjects and Methods Study Population The subjects for this cross-sectional study were enrolled from patients in the Department of Neurology at Qilu hospital between May 2003 and May 2004. The stroke group consisted of 52 patients with first-ever acute hemispheric ischemic stroke of the anterior circulation who underwent MR imaging within 24 hours of the onset of symptoms.8 In addition, 44 asymptomatic patients (asymptomatic group) were also studied who never had symptoms such as amaurosis fugax, transient ischemic attacks, or stroke. At the baseline examination, cardiovascular risk factors including hypertension (blood pressure ⱖ140/90 mm Hg or current use of antihypertensive agents), smoking status (ⱖ10 cigarettes per day for ⬎1 year), and diabetes (fasting blood glucose ⱖ7.0 mmol/L or use of medication for diabetes) were documented and collected through interviews. The following patients were excluded from the study: (1) patients with cerebral hemorrhage, lacunar infarcts, silent infarcts, or posterior circulation infarcts; (2) patients with disorders commonly associated with possible or probable sources of cardioembolism, including atrial fibrillation, cardiac valve abnormalities, recent myocardial infarction, or heart failure; (3) in case of bilateral stenoses, patients with the highest degree of “culprit” carotid stenosis ⬍50%; (4) patients with concurrent diseases or conditions interfering with the expression of inflammatory mediators, such as trauma, surgery or ischemic events during the previous 3 months, severe liver disease, renal failure, hematologic, or malignant diseases, chronic inflammatory diseases, autoimmune diseases, as well as patients with fever or infectious conditions at study entry. The Medical Ethics Committee of Qilu Hospital approved the study, and all patients had to provide written informed consent.
Carotid Ultrasound Examination In symptomatic patients, carotid ultrasound examinations were carried out within 24 hours after onset of symptoms. Ultrasound images were achieved with a 7.5-MHz linear-array transducer (Phillips SONOS 5500). The subject lay in the supine position, with his or her head held in the midline position or slightly tilted to either side. Three different longitudinal views (anterior oblique, lateral, and posterior oblique) and transverse views of both carotid systems were scanned by B-mode and color Doppler methods. Grading of carotid stenosis was determined by routine Doppler criteria as ⬍50%, 50% to 79%, 80% to 99%, or occlusion. Doppler systolic velocity ⬎120 cm/s were used to estimate ⱖ50% stenosis, and a diastolic velocity ⬎135 cm/s indicated ⱖ80% stenosis.9 An atherosclerotic plaque was defined as a focal protrusion 50% greater than the surrounding area and localized thickening ⬎1.2 mm along the extracranial carotid tree. Plaque morphology in terms of echogenicity was graded from 1 to 4 as echolucent, predominantly echolucent, predominantly echogenic, or echogenic, respectively.10 The vessel lumen was used as the reference structure for defining echolucency, and the bright echo zone produced by the media-adventitia interface in the far wall was used as the reference structure for defining echogenicity. Plaque surface structure was classified into 3 categories: smooth, irregular (height variations between 0.4 mm and 2 mm), or ulcerated (ulceration must meet the following criteria: discrete depression ⬎2 mm in width extending into the media, as well as
28
Figure 1. Echolucent plaque with ulceration.
exhibiting an area of reversed flow within the recess or a zone of low flow signal at the level of the recess). In subjects with multiple plaques, only the thickest plaque was selected for analysis. All ultrasound examinations were performed by the same experienced ultrasonographer who was unaware of history and physical findings of the patient, and images of the carotid artery plaques were recorded on magneto-optical discs for further analyses. Representative ultrasound images of atherosclerotic carotid plaques are shown in Figures 1, 2 and 3. To determine the reproducibility of ultrasonic characterization of plaque morphology, a second independent observer reviewed each plaque, and each was blinded to the results of the other. Intra- and interobserver reproducibility of plaque morphology characteristics were greater than 0.80 in our laboratory.
Measurement of Serum Inflammatory Markers Venous blood was collected between 6:00 and 7:00 AM after an overnight fast. Peripheral venous blood was drawn into pyrogenfree blood-collection tubes without any additives and allowed to clot at room temperature for 30 minutes before centrifugation (3500 rpm and 4°C for 15 minutes). Serum was stored at ⫺80°C until analyzed, and the samples were thawed only once. Highsensitivity C-reactive protein (hsCRP) was determined by an ultrasensitive turbidity test based on latex beads (Orion Diagnostica). Serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinases (TIMP-1), and soluble CD40 ligand (sCD40L) were determined with an enzyme-linked immunosorbent assay (MMP-9: R&D Systems; TIMP-1: R&D Systems; sCD40L: Bender MedSystems). Serum levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol were determined using standard automated procedures with commercial kits (Zhejiang dongou technology company, Wenzhou, China). All measurements were done according to the manufacturer’s instructions by laboratory staff unaware of the clinical data.
Figure 2. Predominantly echolucent plaque with ulceration.
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Ding et al
Table 2. Morphological Characteristics of Carotid Plaques
Characteristics
Figure 3. Predominantly echogenic plaque with irregular surface.
Carotid stenosis 50%–79% 80%–99% Echogenicity Echolucent Predominantly echolucent Predominantly echogenic Echogenic Surface irregularity Smooth Irregularity Ulceration
Stroke Group (n ⫽ 52)
Asymptomatic Group (n ⫽ 44)
34 18
30 14
29 10
11 9
6
15
7
9
14 22 16
26 15 3
P 0.772 0.009
0.001
Statistical Analysis All statistical tests have been performed with the SPSS program, version 10.0. Probability values of P ⬍ 0.05 were considered statistically significant. Continuous variables were compared using the Student t test. Categorical variables were compared with the Pearson 2 test.
Results Baseline Clinical Characteristics Table 1 summarizes the age, gender, conventional cardiovascular risk factors, and lipid profiles of participants. Patient characteristics were comparable between the stroke group and the asymptomatic group (P ⬎ 0.05). Ultrasound Analysis of Carotid Plaque Morphology Table 2 shows the results of carotid plaque morphology and carotid stenoses in the stroke and the asymptomatic groups. The distribution of carotid stenosis was similar between stroke patients and asymptomatic patients (P ⬎ 0.05). However, echolucent and predominantly echolucent plaques were more frequently found in stroke patients, whereas
echogenic and predominantly echogenic plaques were seen in asymptomatic patients (P ⬍ 0.05). Smooth plaques were more often observed in asymptomatic patients, whereas irregular and ulcerated plaques occurred more frequently in stroke patients (P ⬍ 0.05). Serum Levels of Inflammatory Markers Table 3 shows the serum levels of inflammatory markers in 2 groups. The serum levels of hsCR, sCD40L, MMP-9 levels were higher in the stroke group than the asymptomatic group (P ⬍ 0.05), whereas serum TIMP-1 levels were higher in the asymptomatic group than in the stroke group (P ⬍ 0.05). Discussion Carotid plaque echogenicity, as assessed by the noninvasive techniques of duplex ultrasonography, is consistent with its histologic characteristics.11 Echolucent plaques are commonly characterized by a large necrotic/lipid core with a thin fibrous cap, whereas echogenic plaques have a higher content of
Table 1. Baseline Characteristics of the Study Participants
Characteristics
Stroke Group (n ⫽ 52)
Asymptomatic Group (n ⫽ 44)
Age (yrs) Male/Females Smokers (%) Diabetes mellitus (%) Hypertension (%) Family history (%) Total cholesterol (mmol/L) Triglycerides (mmol/L) LDL cholesterol (mmol/L) HDL cholesterol (mmol/L)
63.92 ⫾ 9.72 34/18 19 (36.5) 15 (28.8) 30 (57.7) 8 (15.4) 4.91 ⫾ 1.14 1.64 ⫾ 0.95 3.19 ⫾ 0.79 1.07 ⫾ 0.29
61.43 ⫾ 4.39 32/12 18 (40.9) 16 (36.4) 22 (50.0) 6 (13.6) 4.70 ⫾ 1.34 1.84 ⫾ 1.42 3.06 ⫾ 0.90 1.02 ⫾ 0.23
Continuous normally distributed data are shown as mean ⫾ SD. LDL, low-density lipoprotein; HDL, high-density lipoprotein.
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Table 3. Serum Levels of Inflammatory Markers
Characteristics
Stroke Group (n ⫽ 52)
Asymptomatic Group (n ⫽ 44)
hsCRP (mg/L) MMP-9 (ng/mL) TIMP-1 (ng/mL) sCD40L (pg/mL)
5.23 ⫾ 2.18* 260.88 ⫾ 92.80* 58.22 ⫾ 14.95* 10.49 ⫾ 3.56*
1.80 ⫾ 0.34 144.67 ⫾ 34.40 73.48 ⫾ 21.22 7.18 ⫾ 2.77
Continuous normally distributed data are shown as mean ⫾ SD. * Compared with asymptomatic group. hs-CRP, high sensitivity C-reactive protein; MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metalloproteinases; sCD40, soluble CD40 ligand.
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Carotid Plaque Vulnerability and Ischemic Stroke
fibrous tissue and calcification. Echolucent plaques are structurally vulnerable, and several studies have shown that they are associated with an increased risk for ischemic stroke and other cerebrovascular events.12 Gray-Weale et al9 reported that cerebrovascular events were mainly present in patients with either echolucent plaques or echolucent with small echogenic plaques, whereas echogenic plaques with small echolucent areas and plain echogenic plaques were mainly asymptomatic. Grønholdt et al10 reported that echolucent plaques causing ⱖ50% stenosis of the vessel diameter were associated with a higher risk of future stroke in symptomatic but not in asymptomatic individuals. Mathiesen et al13 reported that echolucent atherosclerotic plaques increased the risk of ischemic cerebrovascular events. Recently, Yamashiro et al14 reported that echolucent plaques were more frequently observed in atherothrombotic stroke. In the current study, echolucent and predominantly echolucent carotid plaques were more frequently found in the stroke than in the asymptomatic group of patients. Regarding the significance of the carotid plaque surface, ulceration causes exposure of the thrombogenic lipid core, leading to subsequent intraluminal thrombus formation and, eventually, emboli.15,16 One prospective study17 observed that presence of an irregular plaque was an independent predictor of ischemic stroke risk (adjusted HR, 3.1; 95% CI, 1.1– 8.5). In our study, irregular and ulcerated plaques were detected with greater frequency in the stroke group than in the asymptomatic group. However, other sonographic studies have shown that it was the deep ulceration of plaques and not their surface irregularity, is associated with the presence of symptoms.18 These discordant findings may be related to different definitions of plaque morphology. Disruption of vulnerable carotid plaques, with subsequent arterio-arterial thromboembolism, is an important mechanism of ischemic stroke. However, plaque vulnerability has been shown to be related to the increased local number of inflammatory cells within the plaque, and in particular, to extensive macrophage accumulation.19 MMPs are synthesized and released mainly by activated macrophages, and are capable of degrading all extracellular matrix components. In particular, 92-kDa gelatinase (MMP-9) has been critically associated with carotid plaque vulnerability.20 The activity of these enzymes is tightly controlled by endogenous inhibitors known as TIMPs.21 TIMP-1 is a 28.5kDa glycoprotein that inhibits the activity of MMPs, with a particular affinity for MMP-1, -2, and -9. The degradation of the plaque’s fibrous cap is dependent on the balance between activated MMPs and TIMPs. A recent study by Loftus et al22 indicated a localized increase in the concentration 30
of MMP-9 in the most unstable carotid plaques from symptomatic patients compared with plaques from asymptomatic patients. However, there were no significant differences in the level of TIMP-1 between the 2 groups. Our results were consistent with the findings of Sapienza et al,23 who reported that plasma levels of MMPs were higher in patients with unstable plaques than in patients with stable plaques (P ⬍ 0.001). By contrast, plasma levels of TIMPs were lower in patients with unstable plaques than in patients with stable plaques (P ⬍ 0.001). These results demonstrate that an imbalance between the activities of MMP-9 and TIMP-1 may be responsible for the different clinical manifestations in the stroke group compared with the asymptomatic group. Atherosclerotic plaque rupture itself does not always lead to symptoms, but rather vulnerable blood (prone to thrombosis) with subsequent large thrombus formation plays a key role in the various clinical presentations.24,25 Activation of platelets is a crucial mediator of arterial thrombosis and increased platelet activity has been demonstrated by measuring serum levels of platelet-released products.26 CD40 ligand (CD40L) was found to be expressed on the surface of platelets. It can also be cleaved from cell membranes to form a soluble fragment, sCD40L, which results in the activation of MMPs and procoagulant activity.27 Activated platelets are the major source of sCD40L, and elevated levels of the fragment were reportedly associated with acute coronary syndromes and ischemic stroke.28 In the present study, stroke patients had significantly higher levels of sCD40L compared with asymptomatic patients, suggesting that the increased serum level of sCD40L played a role in platelet activation and atherothrombosis in ischemic stroke. Atherosclerosis is currently recognized as an inflammatory disease, and inflammation has a crucial role in plaque rupture.7,24 C-reactive protein is not only a nonspecific biomarker of systemic inflammation, but also is a contributor to atheroma development, progression, and instability. Serum hsCRP levels provide a means to identify patients at risk for plaque rupture as well as those with a poor prognosis after plaque rupture. Our finding that hsCRP was higher in ischemic stroke than in the asymptomatic patients suggests that systemic inflammation may be responsible for carotid atherosclerosis.29 Study Limitations Our study and the interpretation of our results have several limitations. First, we did not obtain endarterectomy specimens, and serum inflammatory markers were measured only once. Second, other vascular studies, including transcranial Doppler examination, or magnetic resonance angiography, or computed tomography angiography were not carried out to provide further information on the July 2008 Volume 336 Number 1
Ding et al
nature of intracranial large-artery disease. Third, plaque characterization by Doppler ultrasound is usually a subjective process that is observer dependent.30 Finally, this study was cross-sectionally designed and the sample size was small, and further investigation is needed to confirm the clinical significance of the findings.
14. 15.
16.
Conclusions We conclude that structurally vulnerable carotid plaque is closely related to the pathogenesis of ischemic stroke in patients with carotid stenosis, and that inflammation plays a key role in carotid plaque rupture and subsequent atherothrombosis.
17.
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