In the since its initial introduction,1 optical coherence tomography (OCT) has gained prominence as a diagnostic imaging modality in the fields of ophthalmology,2, 3, 4 vascular cardiology,5, 6, 7, 8 and dermatology.9, 10 Major clinical applications of cardiac OCT currently focus on the visualization of the coronary vasculature—for example, identifying unstable plaques and guiding interventional techniques such as stent placement.7 However, within the realms of cardiac research, the applications of OCT have been steadily increasing. Recently, OCT has been used to study developing murine and avian hearts,11, 12 as well as the morphogenesis and myocardial strains of the embryonic chick heart.13 Additional studies have shown the utility of OCT in recognizing components of the cardiac conduction system of the rabbit, specifically the atrioventricular (AV) node14 and the Purkinje network.15 Our recent study extended these findings by exploring the relationship in the rabbit heart between function, visualized through optical mapping, and structure, imaged through OCT.16 Concurrently, tools are also under development to analyze OCT data, including 3-D cardiac fiber structure.17 In this study, we aimed to explore the utility of OCT in identifying components of the conduction system and other structures in the explanted human heart.
Clinical cardiac anatomical visualization and reconstruction techniques, including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound, have a variety of diagnostic capabilities, including localization of arterial plaques, evaluation of bypass grafts and stents, and quantification of wall properties. Although these modalities provide better depth of penetration than OCT, they deliver relatively low resolution images. In biological specimens, OCT, on the other hand, boasts spatial image resolutions ranging from submicrometer through , which can be 10 to 100 times greater than other modalities and comparable to conventional histological analyses.18, 19
In addition to its high spatial resolution, OCT also has other advantages pertaining to diagnostics and surgical guidance. First, OCT can be performed in real time and in situ, allowing for convenient online guidance and evaluation of treatment. Second, OCT technology can be miniaturized to a catheter-based imaging system20, 21 and threaded through the vasculature into the cardiac chambers. Last, OCT can be used for Doppler blood flow and the quantification of blood oxygenation.22, 23
Despite its high spatial resolution, OCT is limited by its depth penetration, estimated to be in nontransparent biological tissues.19 Although there are techniques in development to enhance both its depth penetration and resolution,19, 24, 25 currently standard OCT does not achieve the typical depths of MRI, CT, and ultrasound, which can acquire images of internal structures noninvasively. The advantage of OCT, however, lies in its superior spatial resolution, which enables the visualization of complex fiber architecture, as explored in this study of the explanted human heart.
The use of human hearts for research was approved by the Institutional Review Boards at both Washington University and the University of Minnesota.
Explanted failing human hearts were obtained at the time of cardiac transplantation (Barnes-Jewish Hospital, St. Louis, Missouri) and immediately perfused with cardioplegic solution . Tissue preparations from the right atria (RA), left ventricle (LV), infarcted right ventricle (RV), and atrioventricular junction (AVJ) were isolated from the rest of the heart. An additional sinoatrial nodal (SAN) specimen was obtained through the Upper Midwest Organ Procurement Organization (Life Source, St. Paul, Minnesota) and the University of Minnesota and shipped frozen to St. Louis. The tissues were perfused and fixed with 3.7% formaldehyde overnight and then transferred to 20% sucrose for . The tissues were photographed and clearly marked with pins to precisely correlate the OCT images with histology. OCT was performed later. Table 1 shows the clinical data regarding each specimen.
Clinical data. Relevant clinical information regarding each specimen. The mean age of the patients was 50±10years .
|5||Female||45||Hypertrophic obstructive cardiomyopathy|
OCT, based on the principle of low-coherence interferometry, measures the backscatter of light. The coherence of the backscattered light from the sample arm (or tissue) and reference arm result in the generation of multiple A-scans (axial scans), which are then converted to pixel intensity and combined to form a tomogram (B-scan), or slice of tissue26 (Fig. 1 ). The basics of our OCT system have been described previously.16, 27 We imaged all cardiac tissues in , , or overlapping segments from the epicardial, endocardial, or transmural surfaces, with final axial and lateral resolutions of (in air).
After imaging, the tissue specimens were embedded and frozen in Tissue-Tek OCT compound (Electron Microscopy Sciences, Hatfield, Pennsylvania) and stored at . The tissues were then cryosectioned at , mounted on Superfrost Plus glass slides (Fisher Scientific). Sections were then stained using Masson Trichrome (IMEB, San Marcos, California) and visually compared with the corresponding OCT images.
Computational Analysis and Statistics
Images were processed using MATLAB (Mathworks) and Volocity (Improvision). Initially, each 2-D slice was acquired six times, and the six frames were then averaged together to reduce background noise. Subsequently, during post acquisition data analysis, median filtering was performed with a smoothing kernel to reduce speckle noise.28 Three-dimensional (3-D) tissue reconstructions were created using both Volocity and custom-written MATLAB programs. Additional analysis programs were developed in MATLAB for optical tissue slicing, to determine fiber orientation in relation to the tissue surface,16 and for determination of the depth of OCT penetration. Depth penetration of each field of view acquired across all tissue preparations was assessed via a global thresholding approach.26 Quantitative data are expressed as deviation. A Student’s -test was used to determine statistical significance. A value of was considered statistically significant.
OCT of the Atria and Ventricles
Atrial and ventricular preparations were imaged from both the endocardial and epicardial surfaces. Atrial depth penetration significantly differed from that of the ventricles .
In atrial tissues, we created 3-D reconstructions of the trabeculations and distinguished fiber structures up to below the endocardial surface (Fig. 2 ). In the ventricles, we were able to distinguish changes in fiber orientation in areas located within of each other and below the epicardial tissue surface [Figs. 3a, 3b, 3c ]. Although our current means of imaging does not achieve the depth required to reconstruct fiber architecture throughout the entire thickness of the human myocardium, we do have evidence that this can be accomplished in animal models, in which our current OCT system is able to penetrate through a larger percentage of the myocardium.16
Epicardial OCT can also afford us a view of the coronary vessels. Figure 3d shows a histological cross section through an intact LV coronary vein, with the corresponding OCT cross section shown in Fig. 3e. Figure 4 shows the resultant 2-D and 3-D OCT images from a coronary artery, isolated from the RV epicardial surface, with a main branch diameter of . In particular, note that in Fig. 4c, we are able to distinguish arterial branches less than in diameter, as indicated by the arrowhead.
In addition, we imaged RV infarcts epicardially, endocardially, and transmurally. Although we were able to detect significant changes in depth penetration as compared with adjacent noninfarcted ventricles, as measured from the epicardium and endocardium, we could not visually identify the fibroses from these surfaces with our OCT system. In contrast, when we imaged from the dissected transmural surface, as shown in Fig. 5, the fibrotic tissue of the infarct is clearly visible using OCT and correlates well with histology.
OCT of the SAN
OCT of the SAN was initially attempted from the endocardial surface, the primary point of access during intracardiac procedures. The SAN specimen was dissected to include the RA appendage, crista terminalis (CT), and approximately of atrium beyond the CT [Fig. 6a ]. The specimen was then imaged in overlapping segments, beginning along the CT and extending to the border of dissection. Figure 6b shows 3-D reconstructions of the tissue re-created from scans along the CT/RA border, as indicated by the dashed line in Fig. 6a. Compared with the 2-D tomogram in Fig. 6c, Fig. 6d exhibits an increased number of tissue layers as the transitional area closer to the SAN is approached. As the OCT field of view begins to incorporate the tissue layers above the SAN [Figs. 6e and 6f], it is readily apparent that the primary pacemaker of the heart, defined as a subepicardial structure,29 is located too deep within the myocardium (at ) for imaging from the endocardial surface. The average depth penetration for this specimen from the endocardial surface was . Imaging from the epicardial surface was hindered by a layer of fat along the sulcus terminalis. Although fat intrinsically has a lower backscattered reflectance than normal cardiac tissue,30 the loss in depth penetration was such that we could not visualize the SAN. In addition, the thin layer of epicardial fibrous tissue indicated by the arrow in Fig. 7a may contribute to the loss of depth when the SAN is imaged from the epicardium.
We also imaged the SAN specimen transmurally (i.e., in dissected tissue), which allowed us to consistently locate the SAN throughout its entire length and its proximity to the SAN artery. In this way, we were also able to compare the OCT image to a sister histology section. Due to the prominence of fibroblasts and connective tissue, which serve to isolate the SAN myocytes from the surrounding atrial tissue,31 the SAN is readily identifiable as a unique structure with transmural OCT (Fig. 7), without the need for histological staining.
OCT of the AVJ
OCT of the AVJ was achieved endocardially in five of nine preparations (56%). In the five preparations whose AVJ was imaged, those junctional elements, including the His bundle and AVN, were located within of the endocardial surface. As the average depth penetration in this region across all specimens was , those elements located deeper than , as confirmed via histology, were unable to be imaged in this manner.
All AVJ specimens were dissected for imaging of the entire Triangle of Koch, including a border incorporating additional atrial and ventricular tissues [Fig. 8a ]. The specimens were then optically mapped in overlapping segments, with higher resolution images acquired in regions of interest, particularly the His bundle and atrioventricular node (AVN). In Fig. 8, 3-D reconstructions revealed discrete structures located approximately below the tissue surface [Fig. 8b], corresponding to the locations of the His bundle and AVN as identified with traditional histology. In addition, 2-D tomograms showed this discrete structure encased in fibrous tissue, which manifests as a black envelope surrounding the brighter tissue of the bundle itself [Figs. 8c and 8d]. As our field of view progresses proximal to the His bundle, approaching the rightward extension (RE), also known as the inferior nodal extension (INE), we have a clear view of the AVN/RE [Figs. 8e and 8f]. The clarity is such that we can resolve the blood vessels (ranging from in diameter) located within the AVN itself. The blood vessels are identified due to their consistent locations as identified with both histology and OCT.
Since its inception, OCT has undergone technical advances, including, but not limited to, the establishment of ultrahigh-resolution OCT,32, 33 spectral/Fourier domain OCT,34, 35, 36, 37, 38 numerous improvements in light sources,39, 40, 41 development of a catheter-based imaging probe,20, 21 and ultrasound-enhanced OCT, delivering greater spatial resolution and greater depth penetration.24, 25 However, as OCT technology continues to advance, there still exist technical challenges that remain to be addressed, such as the removal of blood from the imaging field to avoid substantial signal attenuation due to a refractive index mismatch between the cytoplasm of red blood cells (RBCs) and blood plasma. Current intravascular OCT systems incorporate a balloon catheter occlusion system;8 however, this technique cannot be easily applied to imaging the intact heart. In addition, Dextran, a biocompatible agent, has been shown to substantially improve the resolution and depth of OCT imaging through blood by increasing the refractive index of plasma to a value near that of the RBC cytoplasm.42, 43 This technique, known as index matching, has been shown to significantly diminish signal attenuation; however, additional in vivo testing is necessary.
The use of OCT to image the cardiac conduction system and other structures in the intact heart has many potential applications in cardiology. For example, cardiac fibers in the trabeculated endocardial structures of the atria and ventricles, as well as surviving fibers in the infarct border zone, often may contribute to the propensity to develop reentrant arrhythmias.44, 45 Our data indicates that OCT may prove of value in identifying these structures (Figs. 2, 3, 5), and thus potentially arrhythmic areas of the heart.
Although OCT catheters, with diameters as small as , are commercially available for intravascular imaging of vulnerable plaques,8 the size of the target artery and/or tortuosity of the approach may preclude imaging in this manner. In a recent review of the clinical use of intravascular OCT, Guagliumi and Sirbu8 state that cardiac vessels suitable for intravascular OCT are between 2.5 and in diameter and have tortuosity angles less than .8 However, for vessels not exhibiting these characteristics, epicardial imaging may be a viable alternative to intravascular imaging (Figs. 3 and 4).
We have also demonstrated OCT imaging of the conduction system, specifically the SAN and AVJ. These components are vital in the normal function of the heart; therefore, the ability to target them with a high-resolution imaging modality, such as OCT, has any number of potential applications. As the precise location of both the SAN and AVJ within the atrial myocardium is difficult to pinpoint with currently used imaging modalities, their structure can be uniquely identified due to the varying absorption properties present both in the nodal structures, which contain a prominence of fibroblasts and connective tissue, and in the surrounding tissues, as is shown in Figs. 6, 7, 8, using OCT.
Although traditional cardiac imaging modalities, including MRI, CT, and ultrasound, continue to provide physicians with vital information in the diagnosis of cardiac conditions, we have shown the potential of OCT in providing high-resolution, in situ images of the cardiac conduction system and other structures in the intact, explanted human heart. However, despite its ability to distinguish cardiac microstructures as shown in this study, the technique is currently hampered by its limited depth penetration in cardiac tissues. Based on the presented data, we conclude that OCT is emerging as an important imaging modality with the potential to facilitate and guide electrophysiology studies, ablation therapy, implantation of pacing leads, and future cell and tissue engineering therapies subsequent to additional development.
The authors would like to thank Paul Iaizzo (University of Minnesota) for contribution of the sinoatrial node specimen. The authors would also like to acknowledge the following funding sources: NIH R01 HL67322 and AHA Grant-in-Aid (IRE) and NIH DA022871 (CMA).