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Abstract
The ability to evaluate the viscoelastic properties of coronary arteries is crucial for identifying mechanically unstable atherosclerotic plaques. Here, we demonstrate for the first time in living swine, the capability of intravascular laser speckle imaging (ILSI) to measure an index of coronary plaque viscoelasticity, τ, using a human coronary to swine xenograft model. Cardiac motion effects are evaluated by comparing the EKG-non-gated ${\bar{\tau }_{NG}}$, and EKG-gated ${\bar{\tau }_G}$ among different plaque types. Results show that both ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ are significantly lower in necrotic-core plaques compared with stable lesions. Discrete-point pullback measurements demonstrate the capability of ILSI for rapid mechanical characterization of coronary segments under physiological conditions, in-vivo.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Acute coronary events including myocardial infarction (MI) are frequently triggered by the rupture of an unstable atherosclerotic plaque and the subsequent coronary thrombosis [1,2]. Imaging studies in living patients, as well as histopathological examinations in human cadavers have revealed that ruptured necrotic core fibroatheroma (NCFA) plaques are frequently implicated at the sites of coronary thrombi in over 70% of patients who have suffered an MI [3,4]. NCFA plaques are characterized by the presence of a collagen-poor, thin fibrous cap, rich in macrophages, overlying a large lipid pool [3,4]. Reducing the incidence of MI requires identifying and treating these high-risk coronary plaques prior to rupture.
This unmet clinical need has motivated the clinical development of technologies such as optical coherence tomography (OCT), virtual histology intravascular ultrasound (VH-IVUS), and computed tomography (CT), to evaluate key plaque microstructural features such as fibrous cap thickness, plaque burden, and calcific nodules [5–9]. In addition, multi-modal intravascular imaging devices have been developed to simultaneously obtain comprehensive structural and compositional imaging of atherosclerotic plaques, based on combining either IVUS or OCT, with photo-acoustic imaging, near infrared spectroscopy (NIRS), near infrared fluorescence (NIRF), and more recently fluorescence lifetime imaging (FLIM) [10–16]. These novel imaging approaches provide micro-structural images co-registered and colocalized with lipid composition, inflammatory signatures and presence of macrophages, and specific molecular biomarkers related to plaque instability, including lipids, lipoproteins, elastin, collagen, and proteoglycans [10–16].
Compelling evidence, however, suggests that in addition to microstructural and compositional features, plaque rupture is mediated by mechanical factors and in particular, the peak mechanical stress in fibrous cap, which is in turn influenced by the viscoelastic properties of the plaque [17–19]. The presence of a low viscosity necrotic core underlying a mechanically weak fibrous cap depleted of collagen, causes a significant elevation of peak stress, culminating in plaque rupture. Therefore, measurement of the plaque viscoelastic properties is crucial in identifying mechanically unstable plaques with a propensity for rupture and subsequent MI [17–19].
Intravascular Laser Speckle Imaging (ILSI) is a novel optical techniques that affords the unique capability to quantify an index of viscoelasticity of the coronary vasculature [20–23]. Laser speckle is a granular pattern formed by the interference of coherent laser light scattered from tissue [24]. Speckle fluctuations arise from and are modulated by the Brownian motion of endogenous light scatterers and are in turn closely related to the viscoelastic properties of the tissue microenvironment [21,25–30]. Cross correlation analysis of speckle frame series, evolving in time, returns the speckle autocorrelation function, or g2(t). Fitting a single exponential form function to g2(t), provides the speckle decorrelation time constant, τ, which serves as an index of viscoelasticity, for the evaluated plaque [20–23]. Our prior studies conducted in human cadaveric aortas, ex-vivo, and on test phantoms have shown that $\tau $ is closely related to plaque microstructure, composition, and viscoelastic properties [20,21,23,31]. In order to perform ILSI in the coronary vasculature in-vivo, we have developed a flexible, miniaturized optical catheter and demonstrated the capability to quantify arterial viscoelastic properties in living rabbits, as depicted in Fig. 1 [21]. The current manuscript represents the next major stride in the clinical translational journey for ILSI. In this study, we evaluate the capability of ILSI to perform intravascular assessment of human coronary plaques under in-vivo physiological conditions of cardiac and respiratory motion. Using a human coronary to swine xenograft model, we demonstrate for the first time in living swine that ILSI can identify the distinct viscoelastic features of human NCFA plaques under physiological conditions, and therefore may bear the potential for future clinical translation.
Fig. 1. First generation prototype ILSI catheter. (a) The distal end of the catheter, incorporating the single-mode optical fiber (SM600), the GRIN lens, the polarizer and the rod mirror to illuminate the arterial wall (Scale bar: 1 mm). Arterial speckle patterns are collected through the slanted rod mirror, transmitted by a leached fiber optical bundle (SCHOTT, USA; OD 0.7mm; 4.5k fibers; partial core size 0.36), and captured by a high-speed CMOS triggerable camera (Mikrotron, Germany). (b) Custom-fabricated double lumen sheath, housing the ILSI catheter. The inner lumen accommodates the leached fiber bundle and the illumination fiber. The outer lumen incorporates a proximal occlusion balloon (maximum outer dia. 3 mm) and a flushing port to clear the blood from imaging FOV. Reprinted with permission (Ref. [21]).
2. Materials and methods
2.1 Human to swine coronary xenograft model and surgical procedure
To enable ILSI evaluation of human coronary plaque pathology under in-vivo physiological conditions of cardiac motion and coronary luminal blood flow, we selected a human to living swine coronary xenograft model [32]. Using this model, diseased human coronary grafts were surgically sutured to the beating hearts of a living, anesthetized swine and blood flow was re-directed from the swine’s blood circulation to the grafts during ILSI evaluation. The details of the animal procedure are described below and illustrated pictorially in Fig. 2. The animal and human tissue protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Review Board (IRB) at the Massachusetts General Hospital.
Fig. 2. Human coronary to live swine heart xenograft. A median sternotomy was performed to expose the beating heart of the anesthetized swine. The human xenograft was sutured to the anterior wall of the swine heart to simulate physiological motion. An aorto-atrial conduit redirected the blood flow to the grafted coronary via the first inlet of a Y connector. The second inlet allowed for the entrance of the ILSI catheter. A Doppler flowmeter monitored the blood stream redirected through the graft towards the right atrium. Arrow shows direction of blood from aorta (AO) to right atrium (RA). The ILSI catheter was housed in a double lumen sheath. The portable console was comprised of the He-Ne laser source and the bulk optics such as mirrors, beam expander, and fiber coupler (FC) for directing the light into the SMF. The arterial speckle patterns, imaged by the distal optics were transmitted by the fiber bundle to the high speed, triggerable CMOS camera. To generate the trigger signal, the EKG and femoral artery pressure waveforms were fed to a custom-made amplifier module. The amplified signals were digitized and processed by a data acquisition card (NI USB 6251 DAQ) incorporated in the console. The trigger pulse train was fed to the frame grabber for initiating the acquisition in synchrony with the pressure signal (Ref. [21]).
Human cadaveric whole hearts from 3 donors, who succumbed to MI, were obtained from the National Disease Research Interchange (NDRI). The time from autopsy to imaging was between 48 to 72 hours. From each cadaveric whole heart, proximal arterial segments (length ∼5cm) from the right coronary artery (RCA) and left circumflex (LCx) artery were identified, prosected and left partially attached to cardiac flanks, while the vascular side branches were ligated. Discrete arterial locations (n = 26 sites), spaced at ∼0.5cm and indelibly marked with India ink on the outer adventitial surface of these arterial segments were selected for in-vivo ILSI evaluation. These markings were also used to facilitate accurate co-registration of the ILSI evaluated region of interest (ROI) with histopathological analysis. Three Yorkshire swine (50-55 kg) were sedated with Telazol (4.4mg/kg SC), and anaesthetized with isoflurane-O2 gas (1-2% w/3L/min) inhalant while the trachea was intubated and mechanically ventilated. In addition, 180 units/kg of heparin and an antiarrhythmic drug (Amiodarone, 25mg) were also administered intravenously. A median sternotomy was performed to expose the beating heart. The flank of each excised human cadaveric coronary graft, prepared as above, and kept preserved in cold saline at 4°C before implantation, was surgically sutured directly to the anterior wall of the beating heart of each anaesthetized swine. To begin, both ends of the human coronary graft were cannulated with cardioplegia cannulae. On the distal end of the graft, the cannula was inserted into the right atrium chamber of the swine’s heart. On the proximal end, arterial blood from the descending aorta of the swine was re-directed through an infusion pump, to ensure sufficient physiological blood flow rates, and into a Y- introducer attached to the cardioplegia cannula.
The ILSI catheter together with the saline flushing port were also introduced into the graft using the proximal cannula [32]. This set-up is shown in Fig. 2. The blood flow rate was maintained at 30ml/min and monitored with a Doppler flow transducer. During the surgical procedure, the swine’s intra-arterial blood pressure in the femoral artery was continuously monitored and recorded using an in-house blood pressure recording system. The swine EKG signal was monitored and continuously recorded using the custom cardiac data acquisition (DAQ) module interfaced with the portable ILSI console [21].
2.2 ILSI procedure in living swine
The technical details on the fabrication of the ILSI catheter and development of the portable imaging console used in our current study have been previously described in detail [21]. Briefly, the ILSI catheter integrates an inner optical core for illuminating the coronary wall and transmitting the reflected laser speckle patterns to the external high-speed CMOS camera incorporated within the imaging console as shown in Fig. 2. Since the presence of blood affects reflected speckle patterns from of the coronary wall, the ILSI optical core was inserted within a custom-developed catheter sheath that incorporated a proximal occlusion balloon and distal flushing ports for expelling blood from the field of view (FOV) during imaging. The imaging console developed on a portable cart, housed a laser source (632 nm Helium-Neon, randomly polarized, 30mW, JDS Uniphase), and an optical set up to couple light into the catheter to illuminate the arterial wall. A high speed triggerable CMOS camera (MC 1310, 500 fps @ 1280 × 1024 full resolution, dynamic range ∼ 59 dB, Mikrotron, Germany) was incorporated to record laser speckle patterns, ed by the optical catheter, at high acquisition rates (1000 frames per second (fps), 288 × 288 pixel size, ∼59 dB dynamic range). A custom-developed cardiac DAQ module was used to record the swine EKG and/or intra-arterial blood pressure signals during laser speckle pattern acquisition.
To perform the ILSI procedure in-vivo, the ILSI catheter (Fig. 2) was guided into the coronary graft via a proximal catheter introducer and advanced to each discrete imaging location by co-registering the red laser illumination beam visible through the coronary wall with the India ink spot placed on the adventitial surface of the graft. The proximal occlusion balloon was inflated, and each arterial site was evaluated in conjunction with continuous flushing using Lactated Ringers (LR) solution to dispel blood from the FOV during ILSI imaging. To evaluate the influence of cardiac motion on ILSI measurements, the non-gated acquisition of speckle frames, asynchronous to the EKG, was compared to the EKG-gated approach, in which is speckle acquisition is synchronized with the cardiac cycle. This allowed investigating whether the non-gated approach would permit a high-fidelity plaque assessment, similar to that of the gated approach. To this end, at each imaging site, the acquisition of the first speckle image was triggered by the R-wave of the swine EKG signal followed by asynchronous acquisition of subsequent image frames over ∼5 cardiac cycles. An extended acquisition over multiple cardiac cycles was performed purely to permit the comparison between these two approaches, as detailed later. At each marked arterial imaging site, time-varying speckle patterns were acquired at a frame rate of ∼1 kHz over a 500µm FOV on the arterial wall. Following the in-vivo ILSI procedure in living swine, the animals were euthanized, and the coronary grafts were explanted and transferred for histopathological processing (detailed below).
2.3 ILSI data analysis
Computing the speckle intensity temporal decorrelation time constant, τ, which provides the index of tissue viscoelasticity, under both static and deforming conditions has been well described [20–23]. Briefly, laser speckle frame series were obtained and the speckle intensity temporal auto-correlation, g2(t), curves were evaluated using cross-correlation analysis of the first speckle frame with successive frames in the series. For statistical accuracy, both spatial (over the ROI) averaging and temporal (g2(t) curves progressing with time) averaging were conducted. τ was estimated by fitting a single exponential function to the g2(t) curve. Previous studies, conducted ex-vivo, have shown that ILSI still maintains high sensitivity and specificity in determining τ even during ILSI fiber bundle motion or under tissue deforming conditions [20,22,33]. However, motions from both ILSI catheter and coronary wall during the cardiac cycle may influence the ability for ILSI to conduct speckle imaging and provide a measurable τ in-vivo. In order to achieve clinical utility for intracoronary evaluation in patients, the ILSI technology should allow rapid coronary imaging while retaining adequate motion stability over the cardiac cycle. While EKG-gating approaches can be implemented by synchronizing speckle pattern acquisition with the cardiac cycle to mitigate the influence of cardiac motion, this approach adds significant time to the imaging procedure. Instead, a non-gated approach, asynchronous to the EKG is preferred, as it would permit rapid imaging of long coronary segments, which facilitates the use of the ILSI device for intracoronary screening in patients. Therefore, analyses below were performed to investigate the influence of cardiac motion and compare EKG-gated versus EKG-non-gated approaches in conducting ILSI in-vivo.
2.3.1 EKG-non-gated analysis
In the EKG-non-gated approach, analysis of the speckle frame time series was performed by disregarding the fact that the speckle frames were acquired in synchrony with the EKG signal by using a temporal window of 400 sequential speckle frames, randomly selected through choosing arbitrary start points within each of the 5 cardiac cycles. The random starting frame was chosen by a computer algorithm developed and implemented with an in-house Matlab (Mathworks, Natick, MA) code. Subsequently, the speckle intensity temporal auto-correlation curve, g2(t), for each subset of 400 image frames were obtained by computing the normalized cross–correlation of the first frame with each subsequent image in the frame series. Spatial averaging (ROI 288 × 288 pixels) and temporal (averaging window of ∼400ms) were performed. The speckle intensity temporal decorrelation time constant, ${\tau _{NG}}$ corresponding to each plaque type were estimated by fitting a single exponential form function to the initial decay of the g2(t) curve.
2.3.2 EKG-gated analysis
Unlike the non-gated analysis above, in the EKG-gated approach, analysis of the speckle frame time series was performed in synchrony with the EKG signal. That is, the first speckle image frame at the marked imaging site was acquired by progressive triggering of a cardiac gating module in synchrony with the R-wave of the EKG followed by asynchronous acquisition of the remainder of the time varying frames over ∼ 5 cardiac cycles (∼4s). To minimize the impact of cardiac motion on the evaluated speckle auto-correlation time, only a subset of 400 sequential frames, corresponding to ∼ 400ms interval within the resting mid-diastolic phase over each of 5 cardiac cycles of the EKG (∼300 ms from the R-wave) were chosen for analysis [34]. As above, both spatial and temporal averaging were used to evaluate the g2(t) curves for each imaging site. Additionally, the decorrelation time constant, ${\tau _G}$, was computed, by fitting a single exponential form function to the initial decay of the g2(t) curves [20].
2.4 Image and histopathological analysis of arterial tissue type
Following ILSI evaluation, the coronary grafts were fixed in 10% buffered formalin solution for 24 hours, decalcified, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) and Masson trichrome at the location of the fiducial India ink spots. Sections were interpreted by a pathologist (G.J.T) blinded to the ILSI data. In each histology section, plaque tissue type within a 500µm× 500 µm ROI was classified into the following three groups: (1) Necrotic Core Fibroatheroma (NCFA), (2) Fibro-fatty (FA), and (3) Fibrous/Fibro-calcific (FI/FC) [1,3].
2.5 Statistical analysis
From the histopathological evaluation, each plaque was assigned to NCFA, FA, and FI/FC groups. Additionally, for each allocated plaque the corresponding ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ values obtained by ILSI under EKG-non-gated and EKG-gated analyses were recorded. For each of these three groups, the average speckle intensity temporal decorrelation time constant was calculated and expressed as ${\bar{\tau }_{NG}}$ ± SEMNG and ${\bar{\tau }_G}$ ± SEMG, where ${\bar{\tau }_{NG}}$ is the mean time constant for EKG-non-gated analysis, and ${\bar{\tau }_G}$ is the mean time constant of the EKG-gated analysis, and SEMNG and SEMG are the corresponding standard errors of the means. Analysis of variance (ANOVA) was performed to evaluate the capability of ILSI in distinguishing different plaques under both EKG-non-gated and EKG-gated conditions. Additionally, pair-wise multiple comparisons were used to compare every single pair of ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ corresponding to different plaques. The diagnostic threshold level for τ was determined by plotting sensitivity and specificity measurements against the entire range of time constant values. The τ values that accorded with the highest sensitivity and specificity were chosen as the optimal threshold for identifying NCFA from the other two more stable plaque types. Sensitivity, specificity and area under the Receiver Operating Characteristic (ROC) curve were reported with 95% confidence interval (CI). All of the statistical analyses were performed using Prism (GraphPad Inc., San Diego, CA). For all analyses, a probability value, p <0.05 was used to indicate statistical significance.
3. Results
3.1 ILSI measures an index of plaque viscoelasticity
Three out of the 26 plaques obtained from the 26 imaging sites exhibited histological tearing artifacts, likely caused during slicing of the paraffin sections in highly calcific lesions, and therefore were rejected by the pathologist for interpretation and not evaluated in this study. For the remaining 23 arterial sections, the plaque tissue type within the ROI were histologically classified as NCFA (n=4), FA(n=5), and FI/FC (n=14). Figure 3 shows examples of the normalized speckle intensity temporal auto-correlation curves, g2(t), obtained in-vivo in the xenograft swine at 3 coronary graft imaging sites under EKG-gated and EKG-non-gated analysis. As shown in Fig. 3, the NCFA plaque demonstrates rapid speckle intensity temporal decorrelation (${\tau _{NG}}$=1.48ms, ${\tau _G}$=1.84ms) given the low viscosity of lipid. On the other hand, the fibro-fatty (FA) plaque shows a slower speckle intensity decorrelation (${\tau _{NG}}$=9.79 ms, ${\tau _G}$=8.34ms). Lastly, the stiffer fibrous/fibro-calcific (FI/FC) exhibits the slowest g2(t) decay among all (${\tau _{NG}}$=19.58 ms, ${\tau _G}$=29.20ms).
Fig. 3. Speckle intensity autocorrelation curves, g2(t), obtained for three coronary lesion groups, namely Fibrous/Fibrocalcific (FI/FC), Fibro-fatty (FA), and Necrotic Core Fibroatheroma (NCFA), using the EKG-gated and EKG-non-gated ILSI analyses. The g2(t) curves corresponding to different plaque groups are substantially different. At the same time, the g2(t) curves corresponding to the same plaque groups, evaluated using EKG-gated and Non-gated approaches correspond closely.
3.2 Discrete-point ILSI pullback of coronary sections
Figure 4(a) shows the spatial variation of ${\tau _{NG}}$ and ${\tau _G}$ values obtained in-vivo in the human coronary graft as the ILSI catheter is pulled backwards from one imaging site to the next. Clearly, both τNG and τG traces exhibit significant variation, in accordance with the composition and morphology of the atherosclerotic plaques, as evident from the histopathology cross-sections obtained at each site and displayed in Fig. 4(b). In addition, as expected from the statistical analysis above, both curves follow the same trend and reach their minima at the site of an unstable NCFA plaque. More specifically, higher ${\tau _{NG}}$ and ${\tau _G}$ (>20 ms) are observed in the predominantly fibrous regions (<1.5 mm and >3 mm), whereas lower values are seen (<10 ms) in lipid-rich areas (1.5-3 mm). Therefore, the non-gated approach exhibits spatially distinct time constants, τNG, that closely correspond with the spatial variations in plaque composition and morphology in a similar manner to those measured using the EKG-gated approach. For this discrete point measurement of the 3 mm long coronary section (i.e. 7 points), the EKG-gated approach takes up to 14 cardiac cycles, corresponding to 14 seconds. In comparison, the EKG-non-gated approach takes 2.8 seconds. These findings suggest that the time-consuming EKG-gated acquisition and analysis may be conveniently replaced with an efficient EKG-non-gated approach to permit rapid mechanical characterization of long arterial segments, as discussed later.
Fig. 4. (a) The Speckle intensity decorrelation time, τ, evaluated by the ILSI catheter, in-vivo, using both non-gated and gated analysis, as the catheter is manually advanced to discrete arterial imaging sites. (b) The histology image corresponding to each location, along with the plaque type. It is clear that speckle decorrelation time varies in accordance with the arterial wall stability.
3.3 ILSI based plaque characterization
As observed in Fig. 3, the g2(t) curves for the all three plaques, obtained using the EKG-gated and EKG-non-gated analyses have similar trends and closely correspond with each other. Close agreement between the g2(t) curves corresponding to these two different analyses strongly suggests that ILSI presents the capability of being conducted in-vivo without the need for EKG-gating of the speckle signal. The average speckle intensity temporal auto-correlation time constants computed for the three different plaque groups (NCFA, FA, and FI/FC) under EKG-non-gated and EKG-gated analyses are plotted in Fig. 5. The NCFA group exhibits a lower average time constant (${\bar{\tau }_{NG}}$=1.72 ± 0.14, ${\bar{\tau }_G}$=1.88 ± 0.26), compared to the other groups. The FA group presents intermediate average time constants (${\bar{\tau }_{NG}}$=10.55 ± 3.70, ${\bar{\tau }_G}$=13.62 ± 4.23). Finally, the FI/FC group demonstrates the higher average time constants (${\bar{\tau }_{NG}}$=24.07 ± 1.06, ${\bar{\tau }_G}$=24.55 ± 2.09). The overall Analysis of variance (ANOVA), comparing the average time constants among the 3 groups is significant (p< 0.0001). Tukey’s multiple comparison indicate that both ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ are highly significantly lower in NCFA than in FI/FC plaques (p<0.0001 in all 4 cases) (Fig. 5(b)). In addition, both time constants are also significantly lower in FA compared to the FI/FC plaques. Although both ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ are considerably lower in the NCFA compared to FA plaques, these trends are only at the cusp of being statistically significant, likely due to the smaller number of plaques in these two groups. Notably, there are no significant differences between ${\bar{\tau }_G} $ or ${\bar{\tau }_{NG}}$ for either of plaque groups (NCFA, p > 0.99; FA, p = 0.96; FI/FC, p > 0.99), suggesting that ILSI does not require EKG-gating and may instead be performed rapidly and asynchronous with the EKG, by employing fast speckle image acquisition rates and by measuring rapid speckle fluctuations at early decorrelation times.
Fig. 5. (a) The bar diagram representing the average in-vivo speckle decorrelation time constant at both non-gated random cardiac and gated mid-diastole phases, i.e.$\; {\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ for FI/FC, FA, and NCFA lesions. Error bars represent the standard errors of the means. (b) Analysis of variance and tabulated p-values from the multiple comparison analysis, representing the significance of statistical differences between different pairs of groups. Both ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ are highly significantly lower in vulnerable NCFA compared to the stable FI/FC plaques. While not significant, they are also trending lower in NCFA compared to FA.
3.4 Diagnostic sensitivity and specificity
The sensitivity and specificity of ILSI under EKG-non-gated analysis in identifying NCFA and evaluated at a threshold value of τ = 2.37 ms are both 100% (95% CI for Sensitivity: 51.01%-100%, Specificity: 82.41%-100%) while the area under the ROC curve is 1.0 (95% CI: 1.0-1.0, p<0.002). Similarly, for the EKG-gated analysis evaluated at a threshold value of τ = 4.94 ms, sensitivity and specificity are both 100% (95% CI for Sensitivity: 51.01%-100%, Specificity: 83.18%-100%) and the area under the ROC curve is 1.0 (95% CI: 1.0-1.0, p<0.002). These results further indicate the high diagnostic accuracy of ILSI to detect NCFA plaques even under conditions of physiological motion and demonstrate that high diagnostic accuracy for NCFA detection may be obtained even in the absence of EKG-gated speckle acquisition.
4. Discussion
The capability for a timely detection of unstable NCFA plaques is critical for implementing successful diagnostic and therapeutic strategies that aim to prevent adverse coronary events such as MI. This fundamental need has encouraged the evolution of several imaging techniques [5–13,15]. Unstable plaques have primarily been characterized by their morphology, histology, and composition. Existing techniques such as virtual histology intravascular ultrasound [7,35], computed tomography (CT) [8], and optical coherence tomography (OCT) [6,36], have been instrumental in identifying certain morphological distinguishing features of unstable plaques, including fibrous cap thickness, plaque burden, and calcific nodules. In addition, photo-acoustic imaging [11], near infrared spectroscopy (NIRS) [10], near infrared fluorescence (NIRF) [12], and more recently fluorescence life time imaging (FLIm) [13–16] have been invaluable for measuring plaque biochemical and inflammatory signatures, which are key to predicting plaque vulnerability.
A formidable challenge, however, in identifying plaques with the highest risk of rupture in patients is that plaques with comparable morphologic features do not all possess an equal likelihood of rupture. For instance, in 70% of patients dying from MI, multiple thin cap fibroatheromas (TCFAs) were discovered intact, at sites both remote to the culprit plaque and in non-culprit eries [4]. They also appear with similar frequency in stable patients with asymptomatic coronary artery disease (CAD) [4,37–39]. Moreover, in ∼20% of MI cases, plaque rupture was observed in necrotic core (NC) lesions with thicker fibrous caps (>100µm), i.e. thick cap fibroatheroma, in intra-plaque hemorrhages and in calcific nodules [4,38,40,41]. These findings highlight the need to augment morphologic findings with critical surrogate metrics, such as mechanical metrics, in order to accurately identify unstable plaques with the highest risk of rupture [3,4]. The ultimate event of plaque rupture can be considered a biomechanical failure that occurs when a plaque with severely compromised mechanical properties is unable to withstand loads exerted on it [17–19,42]. Therefore, in order to accurately identify plaques with the highest risk of rupture, it is essential to complement morphological and compositional information provided by current technologies with knowledge of plaque mechanical properties.
Here, we demonstrated the significant capability of ILSI for furnishing information on the viscoelastic properties of coronary atherosclerotic plaques in-vivo under physiological conditions of cardiac and respiratory motion and blood flow. We achieved this by utilizing a human diseased coronary to living swine xenograft model that recapitulated both physiological motion and blood flow in-vivo. This sophisticated experimental setting provided a realistic emulation of the actual physiological condition, required for testing the performance of the ILSI catheter on human coronary arteries and enabled investigating key questions on the clinical feasibility of the ILSI technique, as an effective in-vivo diagnostic tool for pinpointing vulnerable plaques in patients.
The first question concerned the ability of the ILS technique to minimize the influence of motion artefacts during the cardiac cycle. To assess the dynamic effects of the cardiac motion on τ, we utilized an EKG-gated technique, in which the speckle frame acquisition at each site was synchronously triggered with the R wave of the EKG signal. Subsequent image frames in the series were acquired asynchronously, over several cardiac cycles. We also evaluated τ in an EKG-non-gated manner previously described above. Intracoronary screening of discrete arterial regions using ILSI was conducted and at each individual imaging site τ values were measured using both EKG-gated and EKG-non-gated approaches. ANOVA and multiple comparison analyses revealed that both the ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G} $ of the NCFA group (${\bar{\tau }_{NG}}$=1.72 ± 0.14, ${\bar{\tau }_G}$=1.88 ± 0.26) were statistically significantly (p<0.0001) than the FI/FC group (${\bar{\tau }_{NG}}$=24.07 ± 1.06, ${\bar{\tau }_G}$=24.55 ± 2.09). Moreover, they were at the cusp of being significant and clearly trending lower compared to FA plaques (${\bar{\tau }_{NG}}$=10.55 ± 3.70, ${\bar{\tau }_G}$=13.62 ± 4.23). We believe that the lack of significance is caused by the relative paucity of NCFA (N=4) and FA (N=5) plaques in our dataset. Increasing the number of plaques in each category in future studies will likely confirm the observed trends in FA vs NCFA time constants in this study. Nevertheless, our findings affirm that ILSI is fully capable of distinguishing the most clinically relevant NCFA plaques which also have a greater propensity for rupturing, from the FI/FC and FA plaques with high sensitivity (100%) and specificity (100%) based on their viscoelastic properties.
The second consideration was the ability of ILSI for rapid spatial screening of long coronary segments. From the tabulated significance levels in Fig. 5(b), we concluded that the statistical differences between ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G} $ were insignificant for all three plaque types (FI/FC, p>0.99; FA, p=0.96; NCFA, p>0.99) . This suggests that we can utilize an EKG-non-gated approach in identifying the vulnerable NCFA plaques as we extend this technology to clinical translation. This will also potentially allow us to image longer coronary segments over a shorter duration, as evidenced by the discrete-point pullback results of Fig. 4, which speaks to the close agreement between the ${\bar{\tau }_{NG}}$ and ${\bar{\tau }_G}$ traces. The success of EKG-non-gated analyses was made possible due to the high-speed speckle acquisition rate (1 kHz) in this study. The high frame rate acquisition enabled capturing the rapid speckle decorrelation times (i.e. τ) of less than 30 ms, elicited by the Brownian dynamics of scattering particles. In our studies, the fitting duration used to obtain the single exponential fit ranged from 25 ms for NCFA plaques to 100 ms for FI/FC plaques. Fitting the exponential form function to the initial decay of g2(t) curves was essential for evaluating the short time-scale dynamics. Given that heart rate occurs at much lower frequencies (i.e. 1–2 Hz) compared with the much higher frequency range of plaque Brownian motion (τ: 1-40 ms, corresponding to 25-1000 Hz), the combination of high-speed acquisition and short-time scale fitting effectively provided stability against the cardiac motions [43]. Since respiration happened at even slower frequencies (0.15-0.5 Hz), its influence was negligible in this study [43].
In the current study, an arbitrary measurement window of 400 speckle frames (400 ms) was used and g2(t) curves were derived. For the non-gated analysis, the 400 ms window was selected at any random location of the cardiac cycle, while for the EKG-gated approach the 400 ms window was selected specifically within the diastolic phase of the EKG to minimize cardiac wall motion. Although the duration of 400 ms was arbitrarily used here, in principle, the measurement duration for g2(t) analysis needs only to cover the initial decay of the g2(t), i.e. fitting duration, which is just slightly longer than the plaque time constant. Therefore, the fitting ranges of 25-100 ms stated above, suggest that in future studies the acquisition time of a single ILSI measurement may be further reduced to ∼100 ms to sufficiently permit the full range of plaque time constants in a rapid intracoronary assessment.
Finally, we tested the capability of ILSI to obtain accurate and diagnostic quality information about plaque viscoelastic properties in the presence of physiological blood flow. In the current study, blood flow was redirected from the native ascending aorta of the swine to the right atrium of the swine heart via the human coronary graft. An infusion pump and flowmeter were incorporated in the path to achieve and confirm a flow rate of 30mL/min which recapitulated typical physiological coronary flow rates in human patients [44,45]. Our prior ex-vivo studies on arterial plaques have shown that the light scattering properties of blood does not affect ILSI process at hematocrit <0.1%. For the in-vivo procedure here, we continuously flushed the vessel using a saline/contrast medium mixture (0.9% saline/Ultravist; conc. 370mg/ml) to fully displace blood from FOV in conjunction with gentle balloon occlusion. Although it was impossible to quantify the intracoronary hematocrit level during the procedure at the imaging site, our results demonstrate the capability to conduct ILSI by temporarily occluding blood flow for short durations during the procedure. Flushing the blood and balloon inflation to obstruct intravascular blood flow present efficient practices for clearing the optical field of view and eliminating the strong blood speckle, which are routinely performed in patients as noted in several other intravascular optical imaging techniques, including OCT and NIRS [46–48]. A safer alternative is to only flush the coronary segments with contrast agents including Visipaque and LR solution [49]. We have conducted early pilot studies in native hearts of living swine to compare coronary wall time constants measured in conjunction with balloon occlusion and coronary flushing. The initial results of these pilot studies suggest that ILSI may be conducted by only flushing the coronaries to clear the blood during imaging and may not require balloon occlusion. Further in-depth studies to compare the balloon occlusion and flushing scenarios are required to verify this possibility.
In this study, imaging was performed with the 1.5 mm outside diameter (O.D.), side-viewing catheter at discrete points purely to test the capability of ILSI to evaluate human atherosclerotic disease in living swine. In this side-viewing catheter, a supple leached fiber bundle, a single mode illumination fiber, and miniaturized distal optics were housed within a pliable double lumen sheath, yielding a highly flexible and mechanically compatible probe that can be safely maneuvered through larger coronary arteries [21]. In addition, the distal end incorporated a soft tip, guidewire port that is similar to that used in clinical grade IVUS or OCT catheters. The soft distal tip ensured that the pull-back could be conducted safely without the risk of puncturing the coronary wall [21]. However, the large size of this catheter may hinder its guidance through small or highly stenotic arteries, rendering it unsuitable for human use. The overall size of the catheter here, was primarily driven by the commercially available optical fiber bundle (dia. 700 µm, ∼4500 optical cores) used in the current design. In another recent study, we have introduced a novel omni-directional catheter that is further miniaturized for imaging smaller coronaries [50]. The optimized, newly designed optical core incorporates multiple illumination fibers, a custom-fabricated smaller fiber bundle (550 µm dia. 3500 optical cores), and an in-house 800 µm dia. multi-faceted pyramidal mirror (MFPM), all within a catheter housing of 1.2mm (O.D.) or 3.6Fr. These size specifications are comparable to the state-of-the-art IVUS catheters (OPTICROSS, 3.6 Fr, 40 MHz, Boston Scientific, Minneapolis, MN), currently used in clinical practice [51]. The new design further affords the capability to scan the entire circumference of the coronary wall in one shot without the need for rotational motion of the catheter [52]. Thus, using a linear pullback stage to retract the probe during imaging, the new catheter may be safely and rapidly guided through long coronary sections in patients, without the need for complex catheter rotation and steering mechanisms, which further enhances the safety and mechanical compatibility of the ILSI probes [52].
While the omni-directional catheter offers a major advantage for future clinical use, in the current in-vivo study, we utilized the side-viewing catheter since it accommodated a more straightforward and precise experimental approach for accurate comparison with histopathological findings within the xenograft swine. More specifically, the side-viewing catheter only illuminated and imaged a single luminal arc angle of the coronary at a time. This allowed precision marking of the catheter location and imaging window, through accurately placing the fiducial ink marks during the in-vivo procedure for subsequent histopathological coregistration and comparison. In contrast, omni-directional viewing made it extremely challenging to accurately mark the circumference of the artery, with respect to the position of the illumination fibers and MFPM facets. We have recently validated the performance of the omni-directional catheter in meticulously designed luminal phantoms, that exhibit distinct viscoelastic properties, along the length, and across 4 lumen quadrants [50]. Future studies entail in-depth validation studies to test the efficacy of the omni-directional catheter in explanted hearts ex vivo prior to performing in-vivo testing.
A key limitation in the current study is the small number of atherosclerotic lesions studied. Given the complexity of the xenograft surgical procedure, only two human coronary segments were grafted in each swine. Thus, a total of 26 plaques from 6 coronary segments were evaluated in the study. Given the small number of plaques, we utilized a broad histopathological classification scheme based on 3 groups – NCFA, FA and FI/FC. The reduced granularly of this plaque typing approach may have increased the standard deviation of the τNG and τG within each group and reduced the significance of the multiple comparison analysis, for instance between NCFA and FA plaques. In particular, the NCFA group includes plaques from a range of fibrous cap thickness values, and FA refers to a large subset of plaques with different populations of adipocytes, foam cells, and macrophages [53]. Similarly, since there were only two fibro-calcific plaques in our cohort, this subtype was grouped with fibrous plaques. While these two plaques did exhibit larger τNG and τG values, our sample size was not powered enough to make significant statistical inferences between FC and FI groups. Future work will involve a larger study with additional donor hearts and swine. Although plaques can be grouped based on their specific signature features as conducted in this study, they may also be highly heterogeneous, showing major attributes of different tissue type groups around the lumen circumference and along its length. Since a side-viewing catheter was employed to measure discrete points along the coronary segment, tissue heterogeneity within the plaque was not accommodated in the study. The omni-directional catheter we have developed addresses this limitation by providing contiguous circumferential viewing of the coronary wall to map heterogenous time constants measured along the circumference and length of the artery [50].
The encouraging results of this study imply that the ILSI may be utilized in an EKG-non-gated fashion, with negligible speckle signal distortion, thus allowing for rapid screening of coronary tissue, under physiological conditions, in-vivo. These studies represent a major validation step in moving ILSI towards clinical translation for patient use. We anticipate in the future that ILSI may be applied in clinical interventional cardiology practice for identifying high-risk plaques and guiding plaque stabilization therapies, either as a standalone approach or as a multimodal outfit, combined with other complementary intravascular imaging techniques such as OCT, IVUS or NIRS.
Over the past few years, a number of speckle-based technological nuances have been developed for the mechanical characterization of various soft tissues and biological fluids [26,27,30,50]. These efforts entail major advances towards precise quantification of the dynamic shear viscoelastic modulus of tissue from the speckle signal, and offer tremendous opportunities for in-vitro diagnosis of coagulation disorders and prediction of thrombotic events within the lab setting [27,29,54–57]. They also afford advanced microscopy platforms, for high-resolution quantification of spatially varying viscoelastic properties, for investigating the pathogenesis and progression of fibrotic diseases and tumor malignancies, in biopsied lesions [30]. The feasibility for tissue mechanical assessment using ILSI in-vivo marks a critical milestone for these speckle-based mechanical characterization technologies, at large, by opening new avenues for the diagnosis of thromboembolic conditions, in both coronary and peripheral arteries, in-vivo. It also paves the path for biomechanical evaluation of other luminal organs via small diameter endoscopes, and other tissue via small-bore needle probes, opening new avenues for imaging in deep inaccessible tissues for research and clinical investigations [50].
Funding
National Institutes of Health (ARRA R21 HL 088306-02S1, R21 HL 088306).
Acknowledgments
We thank Prof. Guillermo J. Tearney for reviewing the histology slides and providing the pathological diagnosis. We also thank Adam Mauskapf for his assistance in conducting in-vivo animal studies. This work was supported in part by the NIH Grant Nos. R21 HL 088306 (S.N) and the ARRA R21 HL 088306-02S1 (S.N.).
Disclosures
ZH: Coalesenz Inc. (I, P) , SKN: Coalesenz Inc. (I, P, S).
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