Collagen and SMCs play a key role in determining plaque stability. Pathologic studies demonstrate that the site of a thrombosed plaque often shows a fibrous cap with diminished collagen content (23
). The overexpression of collagenases alters the mechanical properties further by yielding thinner, disorganized collagen fibers (8
). Smooth muscle cells in plaques synthesize collagen, and their migration and proliferation from the media to the site of an intimal lesion is associated with a net increase in collagen, consequently stabilizing the plaque (5
). The depletion of intimal SMCs may predispose a plaque to rupture, and plaques associated with unstable angina show increased SMC apoptosis (4
In this study, we measured tissue birefringence from PSOCT images of human atherosclerotic plaques. The PSOCT birefringence, Φ, was highly related to total collagen content in all atherosclerotic plaques, as well as in fibrous caps of NCFAs. Φ also demonstrated a strong positive correlation with thick collagen fiber content and a negative correlation with thin collagen fiber content. The inverse relationship between Φ and thin collagen fibers may be explained by the replacement of highly birefringent, thick organized fibers by thin, more randomly oriented fibers. In support of this hypothesis, we found an inverse relationship between thick and thin collagen fibers. Studies have shown that PSOCT measurements of birefringence are influenced by collagen fiber organization; Φ is low when collagen fibers are less organized and high when fibers are well organized (27
). Recent work in animals suggests that thicker collagen fibers in intimal lesions are more aligned and circumferentially oriented than thinner collagen fibers, which show increased structural disorganization (8
). This difference in fiber organization of thick and thin collagen fibers is, therefore, a plausible explanation for our findings that PSOCT birefringence was related positively with thick collagen content and inversely with thin collagen fiber content. We also found that fibrous cap thickness in NCFAs did not bear a statistically significant relationship with PSOCT birefringence (p = 0.81), suggesting that the birefringence measured from PSOCT images may be independent of fibrous cap thickness.
Birefringence exhibited by intimal SMCs may be attributed to their structural construct, comprising a myosin containing thick filament co-assembled with an actin-containing thin filament (28
). The results of our multiple regression analysis showed that when considered jointly with collagen content, SMC content did not demonstrate a significant relationship with Φ. Because plaques in our dataset contained both collagen and SMCs, and because of the strong positive correlation of thick collagen fibers with Φ, it is possible that the variable SMC content dropped out of the multiple regression model. However, when plaques with low collagen content were analyzed separately, Φ showed high positive correlation with SMC content, demonstrating that PSOCT measures birefringence exhibited by SMCs. Previous work has shown that the normalized standard deviation calculated within OCT images is highly correlated with macrophage content (29
). In a separate analysis, we found a statistically significant inverse correlation (r = −0.6, p < 0.005) between the normalized standard deviation computed from OCT images within fibrous caps of NCFAs and Φ in this study. This inverse relationship may be explained by the possible depletion of collagen by metalloproteinases associated with increased numbers of macrophages. In support of this hypothesis, we found a similar inverse relationship between cap collagen content and OCT-normalized standard deviation (r = 0.56, p < 0.01). Taken together, our results indicate that PSOCT birefringence may provide a powerful new index related to plaque stability. Because increased birefringence was correlated to abundant thick collagen fibers and/or SMCs, the detection of high birefringence in PSOCT images may imply increased plaque stability. Conversely, low PSOCT birefringence may indicate compromised plaque stability owing to low collagen content, fewer thick collagen fibers, and/or reduced numbers of SMCs. In all images, phase retardation angles were averaged over the ROI to reduce measurement uncertainty at each depth and facilitate more accurate linear fits for measuring Φ. We observed a significant reduction in statistical uncertainty of depth-resolved phase retardation angles by averaging over 500 µm.
Previous studies have shown that OCT measures multiple factors contributing to plaque instability, including detecting NCFAs, measuring microstructural details such as thin fibrous caps, and identifying cholesterol crystals and quantifying macrophage content (10
). Polarization-sensitive OCT images are always obtained simultaneously with conventional OCT images, providing additional measurements of birefringence related to collagen and SMC content. Thus, OCT enhanced with the capability for PSOCT imaging can identify multiple factors associated with plaque rupture to provide a more comprehensive understanding of plaque stability.
The effects of arterial pulsation on PSOCT measurements have not been analyzed here and warrant further investigation. However, on the basis of prior clinical studies in patients, we have observed that plaque birefringence is preserved during coronary pulsation. New technology has been developed to enable high-speed PSOCT imaging for clinical applications in the near future (30
). Intimal plaque composition of aortas is quite similar to that of coronaries; however, there are significant differences in the media for the 2 arterial types. For this reason, we selected our ROIs to ensure that they were contained only within the intima, and we anticipate that our results can be generalized to coronary lesions. To minimize the influence of tissue shrinkage attributable to formaldehyde fixation and histologic processing (31
) on the data, collagen and SMC content in histologic sections were measured as a percentage of the total area of the ROI. Registration of PSOCT images with corresponding histology sections was performed using fiducial ink marks at the lesion site applied on the plaque using a 26-gauge needle (450 µm); hence, we anticipate that the registration precision between PSOCT and histology was ~500 µm.
Cholesterol crystals, which appear as linear signal-rich regions, may offer another source of birefringence in PSOCT images (). In OCT images, cholesterol crystals can be easily distinguished from other plaque components by their linear and highly reflecting appearance. Three FC plaques in our analysis contained calcific nodules beneath the 200 µm deep ROI, and these regions of calcification were not included in the analysis. Calcific nodules did not contain enough signal for PSOCT measurements, and, therefore, the birefringence of calcifications could not be established. However, calcific nodules can be easily detected with high sensitivity in OCT images as sharply delineated regions within the plaque having signal-poor interiors (17
). Likewise, birefringence in the necrotic cores of NCFAs could not be measured using PSOCT. When light enters the core, because of large variance in scattering structures in the necrotic debris the polarization state of light becomes randomized after multiple scattering events, resulting in unreliable phase retardation measurements (32
) (). Deep within the lipid pool, the signal is greatly attenuated, and PSOCT measurements of birefringence cannot be obtained.
A recent study (13
) has demonstrated a qualitative assessment of plaque collagen using OCT by displaying resulting changes in backreflected light achieved by manually altering the incident polarization state. The PSOCT system used in our current study provides a quantitative evaluation of plaque birefringence, Φ, by measuring the accumulated phase retardation as light travels through birefringent tissue. Our current system measures Φ independent of the incident polarization state of light and sample orientation, thus facilitating intracoronary imaging in patients using catheters similar to those used in conventional intracoronary OCT (9
). A previous feasibility study has demonstrated intracoronary PSOCT in ex vivo coronary arteries using rotary scanning fiber-optic catheters identical to those used in previous clinical trials (33
). The strength of this technique may be further emphasized by noting that PSOCT birefringence is measured in a cross-sectional image, allowing evaluation of discrete microanatomic structures such as fibrous caps. Natural history studies are underway to address questions regarding the role of systemic and local predictors of plaque rupture risk. If the results of these studies show that focal plaque stabilization provides clinical benefits, therapeutic intervention could be guided by information such as that provided by PSOCT, potentially improving patient outcome.
Polarization-sensitive OCT is unique in that it provides images of birefringence, which are co-registered with high-resolution cross-sectional images of plaque morphology obtained by conventional OCT. Beyond the measurement of cap thickness, PSOCT provides additional information about the composition of plaques and NCFA fibrous caps, where low birefringence likely indicates increased instability. Given the potential significance of the additional information provided by PSOCT, and its promise for intracoronary application, we anticipate that this technology will be useful for improving our understanding of the mechanisms of plaque progression and rupture and for the detection of high-risk plaques before the occurrence of an acute coronary event.