Thirty-six nonconsecutive surgical inpatients admitted for carotid endarterectomy for extracranial high-grade (≥ 70%) internal carotid artery stenosis were entered into this study between February 2003 and July 2005 from 3 participating hospitals. Local ethical committee approval was obtained for the study and procurement of specimens. Written informed consent was obtained from all patients. Exclusion criteria were: a disorder that could seriously complicate surgery (3 patients); terminal cancer (1 patient); and patient refusal of operation (1 patient). The study was conducted on 31 common or internal carotid artery plaques from the 31 remaining patients (22 men and 9 women; mean age 68.03 ± 7.3 years). A clinical examination, including neurological exam, with particular care taken to establish the number and duration of ischemic events, and a record of the time from the last symptom and the operation, was obtained from each patient. Before surgery, all patients underwent a: 1 – either cerebral angiography or magnetic resonance angiography and Duplex ultrasound for grading carotid artery stenosis and assessment of intracranial arterial system; and 2 – either computer tomography (CT) or magnetic resonance brain scan. The presence or absence of infarction in the corresponding middle cerebral artery territory was noted. Focal cerebral ischemic events were defined as transient ischemic attack (TIA), amaurosis fugax (AF), central retinal artery occlusion (CRAO), or cerebrovascular accident. Patients were considered to be symptomatic if they had experienced AF, TIA or stroke ipsilateral to the carotid lesion being studied. Silent infarcts and lacunar symptomatology, diagnosed by a neurologist based on clinical and brain computer tomography (CT) scan and/or magnetic resonance imaging (MRI) located ipsilateral to the stenosis, were also considered symptomatic. On the other hand, patients without any history of recent neurologic symptoms or with nonspecific, nonhemispheric symptoms such as dizziness and vertigo were considered asymptomatic. Each patient was then assigned preoperatively to 1 of 2 groups on the basis of their symptom: group I (n = 17; mean age 66 ± 7 years) symptomatic patients; and group II (n = 14; mean age 67.6 ± 6.81 years) consisting of all asymptomatic patients. At the baseline examination, measurements of height, weight, body mass index, blood pressure, fasting serum total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, fasting plasma glucose, electrocardiograms and information about coronary artery disease, diabetes mellitus and smoking habits was collected. Percentages of carotid diameter reduction, procedural methods, concomitant therapy, age, sex, and risk factors did not differ between the 2 groups (Table 1). Nine carotid arteries were removed from human adult cadavers that were not preselected for carotid artery disease. These specimens were used as a control tissue without any macroscopic signs of atherosclerotic plaques.

Carotid plaques were obtained immediately after endarterectomy. All surgeries were performed with standard surgical techniques, and with minimal manipulation of the specimen. No attempts were made to evaluate the presence and the degree of surface ulceration or thrombus. The plaque should be removed in bloc, without fragmentation or significant distortion. After removal, the section of plaque for histological analysis was placed in fresh 4% paraformaldehyde solution and partly decalcified overnight, in order to be sectioned subsequently. The samples were transected transversely at 3 to 4 mm, and embedded in paraffin. For the most of the specimens, five to six blocks were avaiable. Histological analysis was performed by an experience pathologist (SGR), based on American Heart Association classification for human atherosclerotic lesions 34. The inflammatory cell quantification was assessed by light microscopy with final magnification of 400 ×, using an ocular lens with a grid graticule. The observer selected a region in the plaque section with as many inflammatory cells as possible (hot spot). When no inflammatory area was clearly identified the selection was made in an aleatory way. Total area examined was 0.6 mm² and each graticule grid corresponds to an area of 0,0625 mm² with magnification of 400 ×. The inflammatory cells were counted in 10 different graticule area in each specimen. The human carotid arteries removed from cadavers had received the same histological treatment.

Continuous variables were expressed as mean ± SD. Statistical significance was indicated by a value of P < 0.05. The comparison of the histological parameters among the groups was done by non-parametric test of Kruskal-Wallis.

First, the small number of patients was an important study limitation. This limitation will not be easily overcome, since the improvement of carotid artery stenting techniques will make histological analysis of carotid plaques an infrequent procedure. Second, by necessity, the plaques were sectioned and only a small proportion of each plaque was examined microscopically, and it may well be that features were missed in some patients. Most large lesions vary in composition along their length. This may particularly apply to the classification of fibrotic and calcific plaques in group I, where probably different components were missed when a small number of individual sections were examined. Third, we considered in this study patients with lacunar infarctions (LI) as had symptomatic carotid plaques. According to Tejeda et al 8, although significant carotid stenosis was observed at lower levels in LI, its pathogenic value should be taken into account because, when detected on the symptomatic side, it is not only a marker of atheromatosis but also a process potentially linked to LI. And finally, we did not perform imunohistochemical analysis in the present study that would certainly differentiate more instable carotid plaques, with large macrophage infiltration.