Heterogeneities in Cardiac Tissue
4.5 Measuring the Size Distributions
To measure the size distribution in the cardiac coronary vasculature, we acquired µCT data from the atria and ventricles of adult beagle dogs, which were used inin vivo and ex vivo LEAP experiments previously (for more details on these experiments and the µCT reconstruction, see [45]). This also gave us the opportunity to retrospectively relate potential peculiarities in the dynamical behavior during the experiment to the structure of the heart.
4.5.1 Data Acquisition of Dog Heart Vessel Sizes
Five atrial and three ventricular dog heart wedges were scanned [45] in a µCT scanner4at 25 µm voxel resolution in all directions, after injection of 1–2 mL of Microfil5immediately
4GE CT120, GE Healthcare, Little Chalfont, UK
5Flow Tech Inc., Carver, MA
4.5. Measuring the Size Distributions
0.0 0.2 0.4 0.6 0.8
00.050.10.150.20.25Pr(r)
R [mm]
Figure 4.15: The detected “blood vessel” sizes for a phantom made of glass fibers.
The voxel resolution was 60 µm, the glass fibers had nominal radii of 0.25 mm and 0.50 mm.
following theex vivoexperiments, via the same catheter that was used for the perfusion during these experiments. This contrast agent’s radiopacity is based on its lead content;
it is fluid initially, but polymerizes in the hours following administration.
The scanner’s X-ray source was set to 80 kV at 32 µA. From 1200 equally spaced pro-jections over 360° we obtained reconstructed images of 25 and 50 µm voxel resolution.
The volume images were cropped and preprocessed in order to lessen the influence of the catheter which was clearly visible and to reduce the memory requirements. To decrease the random noise of the data, a spatial median filter (over a 3×3×3 voxels cube) was applied as necessary.
I applied the size reconstruction algorithm described in 4.4 to these data sets on high performance computers available at the Max Planck Institute for Dynamics and Self-Organization (16 CPU cores, Intel Xeon E7340 at 2.4 MHz with 4 Mi B cache, 64 Gi B RAM, Linux 2.6.32). The reconstruction procedure took between one and two hours for each data set on these computers.
4.5.2 Porcine Blood Vessel Sizes
To compare the blood vessel size distribution to that in hearts of another species, I adapted the procedure that was used previously in dog hearts and performed supple-mentary size statistic measurements in pig hearts.
4 Heterogeneities in Cardiac Tissue 4.5. Measuring the Size Distributions I preparedN = 7 pig hearts for µCT vasculature scans after they were used in the LEAP experiments described in more detail in the following chapters 6 and 7.
After the end of the experiment, the hearts were first perfused with a hyperkalemic solution to assure a diastolic state and then flushed and covered with 4 % formaldehyde (Roti-Histofix 4 %6) in order to preserve the heart until the scan.
As contrast agent I chose a mixture of 10 % iodo-octane (C8IH177) and 90 % dodecane (C12H267). The iodine content gives enough X-ray contrast to show even small vessels, combined with several advantages over the previously used BaSO4. Since it is a fluid, it will not block the capillaries and there is no risk of sedimentation as could be observed with BaSO4 suspensions during prolonged scans. Additionally we observed several cases of BaSO4 diffusion into the tissue (possibly at fissions of the aortic walls), an effect that should be much less pronounced due to the higher molecular weight of iodo-octane. It shares these properties with the, commercially not available, oil-based contrast agent Angiofil [81].
At least one hour before the µCT scan, the heart was removed from the formaldehyde solution and the formaldehyde in the heart chambers and on the surface was removed.
The coronary vasculature was then manually filled with the contrast agent via catheters leading directly into the left and right coronary arterial branch, respectively. We found that staining the contrast agent with red Sudan III8 helped to apply the correct volume and pressure while filling the vascular system.
The pig hearts were placed in a custom-made acrylic glass container onto low-density foam plastic which was of significantly lower intensity than the water-based heart tissue.
The acrylic glass container also doubled as a “water” phantom of 0 HU9 intensity. In this container, the pig hearts were scanned in aPhoenix NanotomµCT scanner (GE), at 100 kV and approximately 90 µA with 2600 angular steps. Scanning of one sample took approximately nine hours. The scanner images were automatically corrected pixel-wise by their respective dark-image and response to increasing X-ray intensity.
After the scan, the images were corrected for potential movement artifacts during the scan and the volume image was reconstructed at a spatial resolution of around 60 µm.
The contrast agent in the blood vessels had an intensity of up to approximately 1500 HU (see also figure 4.16 for a visualization of the intensity values and figure 4.17 for a three-dimensional rendering).
6Carl Roth, Karlsruhe, Germany
7Sigma-Aldrich, Seelze, Germany
8Sigma-Aldrich, Seelze, Germany
9Hounsfield Units (HU) denote the relative X-ray attenuation of a medium. The Hounsfield scale is normalized so that vacuum has−1000 HU and water has 0 HU.
4.5. Measuring the Size Distributions
0 5 10 15
−100005001500
x [mm]
intensity
Figure 4.16: Cross section through the reconstructed µCT volume of a pig heart.
The intensity of muscle tissue is at around 0 HU, fatty tissue is slightly below. Blood vessels that are filled with contrast agent are at about 1000 HU and above, a value that is comparable to bones. The left ventricle (LV) is at the bottom, the RV at the top, also visible are parts of the atrioventricular valves and of the atria (in the right image half). The intensity values along the white line in the lower right are shown in the plot
on the right hand side.
Figure 4.17: The reconstructed view of a pig heart. Tissue is shown as semi-transparent, the contrast agent is red. The LV is at the right, the RV at the left and the apex at the bottom of the figure. The spatial resolution for the rendered image
is 150 µm, the heart is 83 mm wide.
4 Heterogeneities in Cardiac Tissue 4.6. Electrical Tissue Activation After preprocessing as described for the dog hearts (mostly cutting away unnecessary parts of the volume), the volumes of approximately 15003 voxels were subjected to the same size analysis programs on the Max Planck Institute’s computing cluster. This computation took approximately 9 hours for each data set.