The indication for a CEA is currently largely based on the combination of symptomatology and degree of stenosis (>70%), as defined by flow measurements with duplex ultrasound. These parameters, however, do not provide an accurate assessment of plaque vulnerability and therefore only a small percentage of patients do actually benefit from the surgical intervention by preventing a major cerebrovascular event, especially in the asymptomatic group [20
Indeed, introduction of fluorescent molecular probes, sensitive to plaque vulnerability, has proven to provide a generally good indication of the existence of inflammatory processes. Nonetheless, volumetric fluorescence imaging of plaque activity was so far limited by high degree of scattering in large tissue volumes [9
], which makes it difficult to accurately localize, quantify and characterize the morphology of the plaque and its vulnerability.
In this report, we have shown that MSOT can resolve with high specificity activated proteases, by using the corresponding absorption changes of activatable ‘smart’ probes. It can further deliver high-resolution images of activated probes within tissue, herein an optically scattering human carotid specimen. This newly detected capability was found useful in characterizing plaque formations in atherosclerotic disease. Optoacoustic images can simultaneously show the underlining plaque morphology for accurate localization of MMP activity in three dimensions. Furthermore, the MSOT method can simultaneously provide maps of both activated and inactive probe distribution, thus, provide information on molecular contrast and probe biodistribution without introduction of additional blood pool agent. Our future studies will aim at imaging several different probes optimized for different targets. This could potentially be done by using two probes with peak absorption at say 750 and 680 nm and acquiring multiwavelength optoacoustic data in the spectral windows around 680 and 750 nm. This, however, will certainly require multiple (more than two) wavelengths for efficient spectral unmixing of both probes.
The MSOT findings were verified with a panel of confirmatory studies, i.e., epi-fluorescence imaging of cryosections, zymography and immunohistochemistry. The results from the zymography and immunohistochemistry clearly show a correspondence of MSOT signals and the underlying levels of inflammatory activity, macrophage influx and MMP activity. Results from both MSOT investigations and histological sections confirmed that most of the plaque formation activity occurs, as expected, close to the bifurcation area of the carotid artery.
Even though the current study has dealt with proving the basic feasibility of molecular and morphological characterization of plaques by MSOT, we are currently seeking after in vivo
implementation of the method. Here, the presence of blood will introduce additional absorption of light, which will also become more spectrally dependent. This, in turn, will necessitate more sophisticated light attenuation correction and spectral processing algorithms, which take into account absorption spectra of oxy- and deoxyhemoglobin. Some of those issues could potentially be addressed using blind unmixing methods [23
]. Moreover, even though the current MSOT imaging geometry has been proven successful for whole-body imaging of living mice [24
], clinical translation of the suggested methodology might involve a different technical implementation in terms of light delivery and ultrasonic detection arrangement. This is because most regions of interest in the human body might only be accessible from one side and no full tomographic geometry is available.
Overall, optoacoustics is an inherently three-dimensional visualization tool that has already proven to be capable of penetrating several centimeters into large animals and humans [11
], thus it can potentially image carotid arteries noninvasively. Furthermore, the spatial resolution of the method is not affected by degree of scattering; thus, it can deliver high-quality data from large diffuse tissue volumes, not accessible by other optical imaging techniques. We therefore foresee our MSOT method to be eventually implemented as a noninvasive diagnostic tool for accurate detection and characterization of cardiovascular disease and biomarker activity, e.g., for in vivo
staging of carotid plaque formations for future clinical decision-making.