Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

The preparation of large mammalian hearts using the dehydration and air-drying method removes all water content from the sample. Evidence of insufficient water replacement by ethanol can be observed during HMDS loading (see Protocol, step 4.4). The presence of water under HMDS will create bubbles rising from the tissue. In the case of excessive water levels, a rise in the temperature of the immersion fluid can occur. Keeping the immersion chamber surrounded by ice during initial HMDS loading can reduce the ill-effects of tissue heating. After air-drying hearts in the absence of contrast agents, the sample will appear white in color (see Protocol, step 4.6). The outer surface was often dried and structurally stable before intramural layers. Rinsing in ethanol prior to contrast agent loading removed the white deposit (see Protocol, step 4.7). Slicing through tissue using a sharp blade reveals macroscopically individual muscle fibers with clear separation. Contrast loading by immersing heart samples in contrast agent medium suffered from diffusion limit artifacts in thick and highly muscular regions of the sample. Diffusion contrast loading under vacuum provided more homogeneous coloration in muscle (heart sample #1, see Table 1 for contrast agent loading times). Macroscopically, the surface contrast agent distribution showed in-homogeneous staining between heart muscle and regions composed primarily of extracellular components, notably fat and connective tissue. Air-dried tissue samples, either prior to or after contrast agent loading, maintained stable structural integrity.

The time necessary to scan the full width of the sample at 20 µm resolution under microCT using the abovementioned scanning parameters and an exposure time of 1700 ms was 6 h 34 min. Depending on the size of the sample in the gantry axis of the scanner, this duration was multiplied by the number of positions needed to capture the full length of the specimen. For pig and sheep hearts in this study, three to four positions were used. The NRecon software tiled the multi-position and offset scans to form a single X-ray projection image for each rotation step of the X-ray source and detector. In total, 1000 projections are stored as 16-bit images, generating 30-40 GB of data. Reconstructed volumetric images were 52-70 GB.

Major anatomical landmarks, including the ventricular cavities, septum, and free walls of the ventricles, were easily identifiable from X-ray transmission imaging of air-dried pig hearts stained with contrast agent by diffusion-loading (Figure 1A). Moreover, highly textured regions indicating microstructural organization, such as myocardial fiber orientation, were also observed due to sensitive X-ray attenuation/transmission (Figure 1B). Tomographic reconstructions of three-dimensional image volumes showed distinct separation between tissue and background at both epicardial and endothelial boundaries (Figure 1D). Intramurally, low contrast and voxel intensity diffusion gradient were observed throughout thick transmural regions of the tissue. Despite that, vasculature and myocardial fibers separated by cleavage planes were still readily identifiable. A second higher intensity bandwidth of contrast was observed at the epicardial-most layer and in punctate sub-endocardial regions. Contrast enhancement was greatest at sites where extracellular components were accumulated, particularly epicardial connective tissue, epicardial fat and the connective tissue sheath of the Purkinje fiber network. Voxel signal intensity distributions showed high separation from the zero-intensity background (air) and two dominant populations of low and high contrast tissue (Figure 1D).

To validate contrast enhancement of microCT image reconstructions and the selectivity to collagenous compartments of the heart samples, histology, bright field microscopy, and fluorescent microscopy were employed (Figure 2). A transmural block of ventricular tissue from an air-dried heart without prior contrast agent-loading was prepared for paraffin embedding and sectioning. Adjacent tissue slices mounted on microscope slides were treated by either Masson's trichrome staining, no treatment, or 48 h of PMA (1%). Immersion of slide-mounted tissue sections eliminated diffusion gradient effects of the staining process that was observed in whole heart samples. Mason's trichrome staining showed collagen-positive staining at the epithelial and endothelial layers, perivascularly in the sub-epicardial tissue, and a connective tissue sheath surrounding a free-running Purkinje fiber protruding into the left ventricular cavity (Figure 2A). Bright field illumination showed darker coloration in collagenous structures after PMA-staining, supporting the preferential accumulation of PMA (Figures 2B,C). Moreover, PMA treatment has previously been shown to quench the autofluorescence of collagen macromolecular complexes³¹. Fluorescent images of ventricular tissue sections had PMA-induced loss of fluorescence at sites of collagen (Figure 2D vs. 2E, Figure 2D' vs. 2E' and Figure 2D'' vs. 2E''). In both bright field and fluorescent imaging, cellular compartments were not altered by the PMA treatment, and collagen had a selective accumulation of PMA staining and quenching of autofluorescence.

Heart sample #2 was stained with a contrast agent via perfusion prior to air drying. Image reconstruction revealed highly patchy staining within the myocardial compartment (Figure 3A). Contrast enhancement appeared unselective of tissue composition, with no further enhancement of signal intensity at the epicardial or sub-endocardial regions. Moreover, low contrast tissue showed poor separation from the background intensity (Figure 3B).

Ventricular fibrosis was induced by myocardial infarction and chronic ischemia (Heart sample #3). An antero-apical scar was formed by replacing myocytes with fibro-fatty deposits in the tissue downstream to the site of vascular embolization. Heart sample #3 was prepared and imaged from a dissected ventricular wedge covering the anterior left ventricle, septum, and right ventricular free wall. The preparation of this ventricular wedge configuration has been described previously³² and the application of wedges for cardiac imaging was reviewed in detail³³. Scar morphology was transmural but heterogeneous (Figure 4). A central dense fibrotic lesion was surrounded by a loose and heterogeneous border zone (Figure 4A). The ventricular preparation was stained by diffusion-loading post-air drying and in a vacuum. Figure 4B–E shows the greatest signal intensities of reconstructed microCT image volumes at the tissue boundaries and scar regions. Contrast agents poorly stained healthy myocardium, yet microstructural contrast was retained (Figure 4C'). At the border zone, scar tissue was interspersed with surviving myocardium (Figure 4D'). Dense fibrosis appeared transmural yet textured, indicating variances in composition (Figure 4E'). Tissue sections of a transmural left ventricular region of the air-dried and PMA-stained tissue preparation were used to validate PMA selectivity for collagen in pathological tissue by comparing against Masson's trichrome staining (Figure 4F). PMA staining was selective for collagen (sub-epicardium and sub-endocardium) and absent in regions of surviving myocardium (Figure 4G).

Heart sample #4 with induced persistent atrial fibrillation was air-dried while conserving the native shape of the atrial cavity. Atrial appendage collapse was not observed. The major anatomical landmarks could be identified morphologically from reconstructed images (the atrial septum, pectinate muscles, coronary sinus, pulmonary vein ostia, vena cava and cristae terminalis). Diffusion-staining under vacuum resulted in contrast enhancement in the aortic root and atrioventricular valves and discrete regions of the working myocardium. Muscle staining enhancement was constrained to the atrial appendages and posterior walls of both left and right atria (Figure 5).

Figure 1: MicroCT imaging of an air-dried pig heart treated with PMA contrast agent by diffusion under vacuum. (A) X-ray projection image. (B) A transmission profile extracted from the red line in A. (C) Short-axis slice of the ventricles from a tomographically reconstructed three-dimensional volume. Yellow arrows indicate punctate regions of contrast attributed to sub-endocardial Purkinje fibers. Blue arrows indicate vasculature. (D) Signal intensity distribution of the reconstructed image slice shown in C. LV: left ventricle and RV: right ventricle. Please click here to view a larger version of this figure.

Figure 2: Validation of PMA-selectivity for collagen. (A) Masson's trichrome staining of a transmural tissue section from the ventricles of an air-dried heart. Myocardium is stained in red and collagen is shown with green coloration. Adjacent tissue sections (B) absent of staining or (C) stained with PMA (1%) were imaged with bright field illumination to assess the uniformity of coloration. (D) Tissue sections absent of staining or (E) stained by PMA were imaged by fluorescent microscopy. Panels D' (solid red box) and E' (dashed red box) are enlarged views of the sub-epicardium for unstained and PMA-stained sections. Panels D'' (solid blue box) and E'' (dashed blue box) are corresponding enlarged views of the sub-endocardium and a free-running Purkinje fiber. Arrows indicate sites of collagen content. Please click here to view a larger version of this figure.

Figure 3: Perfusion-loading of PMA prior to air-drying and MicroCT imaging. (A) A short-axis slice of a reconstructed image volume of the ventricles from a pig heart. Blue arrows indicate vasculature. (B) The signal intensity distribution of the image slice from panel A. LV: left ventricle and RV: right ventricle. Please click here to view a larger version of this figure.

Figure 4: MicroCT imaging of a sheep heart suffering a chronic myocardial infarction. (A) A dense scar was formed in the apical region (see inset photograph). A volume rendering of the apical region from an endocardial perspective was assigned coloration based on image intensity (red corresponding to scar tissue and myocardium in green). Orthogonal slices of the greyscale intensity show the dense scar distribution and bordering surviving myocardium. Separation between fibrotic tissue and myocardium corresponds to regions of adipose tissue. (B) A photograph of an air-dried ventricular wedge preparation from a sheep with apical scarring following myocardial infarction. Oblique slices of the reconstructed microCT image volume traverse the ventricles at the mid-level between base and apex and proximal to the site of (C) vascular occlusion (C– red line in panel B), (D) the peri-infarct region bordering dense scar and healthy myocardium (D– blue line in panel B) and (E) a region of dense fibrosis (E – green line in panel B). (C') An expanded view of the septal region outlined by a red dashed box in C. (D') An expanded view of the infarct region in the right ventricular apex (blue dashed box in panel D). (E') An expanded view of the infarct region in the left ventricular apex (green dashed box in panel E). LV: Left ventricular cavity; RV: right ventricular cavity; MB: moderator band; Pap: papillary muscle. Yellow arrow indicates left anterior descending artery. (F) Masson's trichrome staining of a histological section cut from the PMA-stained air-dried left ventricle. Collagen is stained blue and myocardium is stained pink/violet. (G) A corresponding tissue section of the PMA-staining distribution. Please click here to view a larger version of this figure.

Figure 5: MicroCT image of a sheep heart following chronic induced atrial fibrillation. (A) A volume rendering of the atria with coloration assigned as in Figure 4A. (B) Bi-atrial microCT image slice in the long-axis of the heart. Short-axis slices were extracted at the level of the (C) atrioventricular valves (C– red line in panel B), (D) aortic root (D– blue line in panel B) and (E) left atrial roof (E– green line inpanel B). LA: left atria; RA: right atria; LAA: left atrial appendage; RAA: right atrial appendage; LV: left ventricle; RV: right ventricle; LVOT: left ventricular outflow tract; RVOT: right ventricular outflow tract and PA: pulmonary artery. Please click here to view a larger version of this figure.

Table 1: Heart samples and contrast agent treatment.