Characterization of Coronary Arteries: Correlating Mechanical Stiffness with Staining for in vivo Imaging

Abstract

Widespread prevalence of cardiovascular disease (CVD) in the US is indisputable. Over 82.6 million adults (~30% of the US population) have been diagnosed with one or more forms of CVD. Coronary artery disease (CAD) is the most common form and constitutes nearly 50% of all cases. It remains the leading cause of death for individuals afflicted with CVD due to the critical role of the coronaries in myocardial function. In coronary arteries, the CAD manifests as a highly-active, immune-driven sequestration of lipids which eventually triggers calcification that is found in advanced CAD lesions. Calcification is thought to provide stability as an atheroprotective mechanism even though deposition results in gradual stenosis and angina. Unstable or ‘vulnerable’ lesions are characterized with very little calcium deposition, yet these lesions are thought to result in myocardial infarction. Vulnerable lesions are difficult to observe by traditional approaches and are differentiated by thin-cap fibroatheromas (TCFAs). TCFAs are large, eccentric, necrotized lipid cores that are contained within the arterial wall by a thin fibrotic cap. Vulnerable lesions do not occlude the lumen and are not evident under conventional, non- invasive imaging, thus monitoring their structural progression is paramount for improving clinical outcomes. Histopathology is the gold standard for assessing lesion grade through H&E and other more specific stains. These dyes are toxic which preclude their use in vivo interrogation. Several imaging modalities have thus been developed in lieu of conventional histology to circumvent this limitation. One of which is the Scanning Fiber Endoscope (SFE). Pilot SFE investigations on cadaveric specimens and biomarkers between the Human Photonics Lab (HPL) and the Center for CardioVascular Innovation (CCVI) led to the exploration and application of Evan’s blue as a contrast agent for fibrotic caps. Evan’s blue is an FDA-approved compound and is typically employed as an in vivo blood tracer for Boolean assessment of cardiac leakage, or output. The chemical compound also demonstrates increased affinity for fibrous cap components and exhibits spectral characteristics that permit SFE fluorescence imaging. Evan’s blue stains in a graded fashion reflecting fibrotic cap content. An increased fibrotic cap content is directly associated with an increase in mechanical stiffness. We therefore hypothesize that the optical intensity of Evan’s blue correlates with the mechanically stiff properties of diseased coronary arteries to support SFE detection for in vivo assessment. To test our hypothesis, the experimental histology laboratory at the CCVI was tailored to process human and porcine coronary specimens. This included all the classical steps in pathology along with high resolution imaging for quantification. Procured human (n = 7) and porcine (n = 10) specimens were first tested in a custom-designed mechanical apparatus assembled at HPL prior to histology. In proximity to the contracting myocardium, coronaries experience a greater amount of longitudinal stress during physiological activity and when coronaries are diseased. The mechanical apparatus thus measured displacements within specimens when a load (20-200 g) was applied to the sample. Coronary arteries were isolated and segmented (normal: n = 20, diseased: n = 14), and lengths/diameters were precisely measured. Segments were then mounted on a machined probe for testing. Vessel segments were loaded several times (n = 5) for a given mass and the experiment was repeated at least 5-8 times for fresh and frozen groups for both normal and diseased coronary segments. A knot in the suture line attaching the specimen to the calibrated mass provided a fiducial marker for vessel displacement. High resolution, high-speed cameras acquired video recordings of knot displacements where optical data were processed, and analyzed using ImageJ and MATLAB code. Displacement vs. force and stress vs. strain plots were produced for all experiments. Fresh, healthy segments displaced 500 um more than diseased samples and spanned a range of 1.5-3.3x. Frozen, healthy segments displaced an average of 400 um less than fresh segments suggesting the presence of a freezing-induced artifact. Comparisons between human and porcine segments revealed similarities in their displacements suggesting that porcine arteries can substitute for disease-free arteries during longitudinal uniaxial testing. Following mechanical characterization, coronary segments were processed into microscope slides for pathological evaluation. Sections (n > 10 slides/group) were stained (Verhoeff’s Hematoxylin - elastin/calcification, Aniline Blue - collagen, Evan’s Blue - fibrotic caps) and quantified via ImageJ to determine collagen-to-elastin (C/E) ratios and degree of Evan’s Blue staining. Although diseased tissue segments displaced less than normal segments, stress-strain analyses and the resulting Young’s modulus values did not confirm our hypothesis of an expected increase in stiffness. It is thought that the varying cross-sectional areas of diseased segments may have contributed to this effect as a diameters and hence cross-sectional areas between groups were different by nearly 3 fold.