Phosphotungstic acid (PTA) preferentially binds to collagen- rich regions of porcine carotid arteries and human atherosclerotic plaques observed using contrast enhanced micro-computed tomography (CE-µCT).

Background and aims: Atherosclerotic plaque rupture in the carotid artery can cause small emboli to travel to cerebral arteries, causing blockages and preventing blood flow leading to stroke. Contrast enhanced micro computed tomography (CEµCT) using a novel stain, phosphotungstic acid (PTA) can provide insights into the microstructure of the vessel wall and atherosclerotic plaque, and hence their likelihood to rupture. Furthermore, it has been suggested that collagen content and orientation can be related to mechanical integrity. This study aims to build on existing literature and establish a robust and reproducible staining and imaging technique to non-destructively quantify the collagen content within arteries and plaques as an alternative to routine histology. Methods: Porcine carotid arteries and human atherosclerotic plaques were stained with a concentration of 1% PTA staining solution and imaged using MicroCT to establish the in situ architecture of the tissue and measure collagen content. A histological assessment of the collagen content was also performed from picrosirius red (PSR) staining. Results: PTA stained arterial samples highlight the reproducibility of the PTA staining and MicroCT imaging technique used with a quantitative analysis showing a positive correlation between the collagen content measured from CEµCT and histology. Furthermore, collagen-rich areas can be clearly visualised in both the vessel wall and atherosclerotic plaque. 3D reconstruction was also performed showing that different layers of the vessel wall and various atherosclerotic plaque components can be differentiated using Hounsfield Unit (HU) values. Conclusion: The work presented here is unique as it offers a quantitative method of segmenting the vessel wall into its individual components and non-destructively quantifying the collagen content within these tissues, whilst also delivering a visual representation of the fibrous structure using a single contrast agent.

Microstructural and mechanical insight into atherosclerotic plaques: an ex vivo DTI study to better assess plaque vulnerability.

Non-invasive microstructural characterisation has the potential to determine the stability, or lack thereof, of atherosclerotic plaques and ultimately aid in better assessing plaques' risk to rupture. If linked with mechanical characterisation using a clinically relevant imaging technique, mechanically sensitive rupture risk indicators could be possible. This study aims to provide this link-between a clinically relevant imaging technique and mechanical characterisation within human atherosclerotic plaques. Ex vivo diffusion tensor imaging, mechanical testing, and histological analysis were carried out on human carotid atherosclerotic plaques. DTI-derived tractography was found to yield significant mechanical insight into the mechanical properties of more stable and more vulnerable microstructures. Coupled with insights from digital image correlation and histology, specific failure characteristics of different microstructural arrangements furthered this finding. More circumferentially uniform microstructures failed at higher stresses and strains when compared to samples which had multiple microstructures, like those seen in a plaque cap. The novel findings in this study motivate diagnostic measures which use non-invasive characterisation of the underlying microstructure of plaques to determine their vulnerability to rupture.

Diffusion tensor imaging and arterial tissue: establishing the influence of arterial tissue microstructure on fractional anisotropy, mean diffusivity and tractography.

This study investigates diffusion tensor imaging (DTI) for providing microstructural insight into changes in arterial tissue by exploring how cell, collagen and elastin content effect fractional anisotropy (FA), mean diffusivity (MD) and tractography. Five ex vivo porcine carotid artery models (n = 6 each) were compared-native, fixed native, collagen degraded, elastin degraded and decellularised. Vessels were imaged at 7 T using a DTI protocol with b = 0 and 800 s/mm2 and 10 isotopically distributed directions. FA and MD were evaluated in the vessel media and compared across models. FA values measured in native (p < 0.0001), fixed native (p < 0.0001) and collagen degraded (p = 0.0018, p = 0.0016, respectively) were significantly higher than those in elastin degraded and decellularised arteries. Native and fixed native had significantly lower MD values than elastin degraded (p < 0.0001) and decellularised tissue (p = 0.0032, p = 0.0003, respectively). Significantly lower MD was measured in collagen degraded compared with the elastin degraded model (p = 0.0001). Tractography yielded helically arranged tracts for native and collagen degraded vessels only. FA, MD and tractography were found to be highly sensitive to changes in the microstructural composition of arterial tissue, specifically pointing to cell, not collagen, content as the dominant source of the measured anisotropy in the vessel wall.