Objective: Atherosclerotic plaque rupture in carotid arteries is a major source of cerebrovascular events. Calcifications are highly prevalent in carotid plaques, but their role in plaque rupture remains poorly understood. This work studied the morphometric features of calcifications in carotid plaques and their effect on the stress distribution in the fibrous plaque tissue at the calcification interface, as a potential source of plaque rupture and clinical events. Methods: A comprehensive morphometric analysis of 65 histology cross-sections from 16 carotid plaques was performed to identify the morphology (size and shape) and location of plaque calcifications, and the fibrous-tissue fiber organization around them. Calcification-specific finite element models were constructed to examine the fibrous plaque tissue stresses at the calcification interface. Statistical correlation analysis was performed to elucidate the impact of calcification morphology and fibrous tissue organization on interface stresses. Results: Hundred-seventy-one calcifications were identified on the histology cross-sections, which showed great variation in morphology. Four distinct patterns of fiber organization in the plaque tissue were observed around the calcification. They were termed as attached, pushed-aside, encircling and random patterns. The stress analyses showed that calcifications are correlated with high interface stresses, which might be comparable to or even above the plaque strength. The stress levels depended on the calcification morphology and fiber organization. Thicker calcification with a circumferential slender shape, located close to the lumen were correlated most prominently to high interface stresses. Conclusion: Depending on its morphology and the fiber organization around it, a calcification in an atherosclerotic plaque can act as a stress riser and cause high interface stresses. Significance: This study demonstrated the potential of calcifications in atherosclerotic plaques to cause elevated stresses in plaque tissue and provided a biomechanical explanation for the histopathological findings of calcification-associated plaque rupture.

Background: Calcification and inflammation are atherosclerotic plaque compositional biomarkers that have both been linked to stroke risk. The aim of this study was to evaluate their co-existing prevalence in human carotid plaques with respect to plaque phenotype to determine the value of hybrid imaging for the detection of these biomarkers. Methods: Human carotid plaque segments, obtained from endarterectomy, were incubated in [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt), targeting Leukocyte Function-associated Antigen-1 (LFA-1) on leukocytes. By performing SPECT/CT, both inflammation from DANBIRT uptake and calcification from CT imaging were assessed. Plaque phenotype was classified using histology. Results: On a total plaque level, comparable levels of calcification volume existed with different degrees of inflammation and vice versa. On a segment level, an inverse relationship between calcification volume and inflammation was evident in highly calcified segments, which classify as fibrocalcific, stable plaque segments. In contrast, segments with little or no calcification presented with a moderate to high degree of inflammation, often coinciding with the more dangerous fibrous cap atheroma phenotype. Conclusion: Calcification imaging alone can only accurately identify highly calcified, stable, fibrocalcific plaques. To identify high-risk plaques, with little or no calcification, hybrid imaging of calcification and inflammation could provide diagnostic benefit.

Purpose: Atherosclerotic plaque development and progression signifies a complex inflammatory disease mediated by a multitude of proinflammatory leukocyte subsets. Using single photon emission computed tomography (SPECT) coupled with computed tomography (CT), this study tested a new dual-isotope acquisition protocol to assess each radiotracer’s capability to identify plaque phenotype and inflammation levels pertaining to leukocytes expressing leukocyte function-associated antigen-1 (LFA-1) and the leukocyte subset of proinflammatory macrophages expressing somatostatin receptor subtype-2 (SST₂). Individual radiotracer uptake was quantified and the presence of corresponding immunohistological cell markers was assessed. Methods: Human symptomatic carotid plaque segments were obtained from endarterectomy. Segments were incubated in dual-isotope radiotracers [¹¹¹In]In-DOTA-butylamino-NorBIRT ([¹¹¹In]In-Danbirt) and [^99mTc]Tc-[N^0–1 ₄,Asp⁰,Tyr³]-octreotate ([^99mTc]Tc-Demotate 2) before scanning with SPECT/CT. Plaque phenotype was classified as pathological intimal thickening, fibrous cap atheroma or fibrocalcific using histology sections based on distinct morphological characteristics. Plaque segments were subsequently immuno-stained with LFA-1 and SST₂ and quantified in terms of positive area fraction and compared against the corresponding SPECT images. Results: Focal uptake of co-localising dual-radiotracers identified the heterogeneous distribution of inflamed regions in the plaques which co-localised with positive immuno-stained regions of LFA-1 and SST₂. [¹¹¹In]In-Danbirt and [^99mTc]Tc-Demotate 2 uptake demonstrated a significant positive correlation (r = 0.651; p = 0.001). Fibrous cap atheroma plaque phenotype correlated with the highest [¹¹¹In]In-Danbirt and [^99mTc]Tc-Demotate 2 uptake compared with fibrocalcific plaques and pathological intimal thickening phenotypes, in line with the immunohistological analyses. Conclusion: A dual-isotope acquisition protocol permits the imaging of multiple leukocyte subsets and the pro-inflammatory macrophages simultaneously in atherosclerotic plaque tissue. [¹¹¹In]In-Danbirt may have added value for assessing the total inflammation levels in atherosclerotic plaques in addition to classifying plaque phenotype.