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Statistical Analysis of Transmural Laminar
Microarchitecture of the Human Left Ventricle
Iulia Mirea1, Lihui Wang2, François Varray1, Yue-Min Zhu1, E. E. Dávila Serrano1, Isabelle E. Magnin1
1
Lyon University, CREATIS, CNRS, Inserm, Lyon, France
2
Guizhou University, Guiyang, China.
Abstract- A good knowledge of the cardiac microarchitecture
is essential for better understanding the function of the human
heart. This paper investigates the transmural 3D microstructure
of the left ventricle of the human heart. An ex-vivo sample (7 x 7
x 15 mm3) extracted from the anterior wall of the myocardium is
imaged using X-rays phase contrast micro-tomography.
Sampling the volume at high isotropic resolution of 3.5 x 3.5 x 3.5
μm3 allows a clear reveal of the laminar structure of collagen
wrapping the myocytes groups. An image processing protocol is
developed to automatically extract cleavage planes and compute
statistics of their thickness and distance separating them. The
results show the clear presence of cleavage planes in the
myocardium and their variation in terms of thickness, interplanes distances and local orientation, which contribute to a
better understanding of the human heart function.
Keywords— cardiac tissue; 3D; high-resolution; phase contrast
imaging; myocytes sheets; cleavage planes;
I. INTRODUCTION
A good knowledge of the cardiac microarchitecture is
essential for understanding the mechanics of the myocardium
and of the human heart electrical activity, and the relations
between mechanical function, hemodynamics and the adaptive
structural changes in cardiac diseases [1, 2]. For this reason,
detailed information about the organization and the spatial
relationships between the cardiac tissue components is
required. However, our understanding of the cardiac
architecture is limited by the lack of 3D descriptions of the
tissue organization at a small or microscopic scale.
One of the main components that define the 3D myocardial
tissue organization and function is the cardiac extracellular
matrix. The cardiac extracellular matrix includes: endomysium
that surrounds and separates individual myocytes and
capillaries, perimysium that surrounds and separates groups of
myocytes and epimysium that surrounds the entire heart
muscle.
Previous studies demonstrate that the ventricular
myocardium has a laminar organization, where the myocytes
are organized in groups separated by cleavage planes “shear
layers” [3]. The perimysium, a connective tissue matrix made
of collagen, contains the cleavage planes and various
components as blood vessels, capillaries, lymphatic vessels.
The 3D arrangement of myocardial lamellae accommodates the
changing myocytes orientations across the ventricular wall and
plays an important role in the mechanical activity of the heart
[4, 5, 6, 7].
The description of the transmural architecture has been
achieved using 2D techniques such as optical images of
histological sections. Such techniques provide high 2D spatial
resolutions but suffer from distortions due to cutting thin
samples [5]. Although these techniques yield important 2D
morphological information, they are not fully adapted to
reconstruct complex 3D microstructures that can be resliced
and reoriented with much more details. 3D techniques such as
polarized light microscopy, transmission or scanning electron
microscopy (TEM, SEM) [1, 8, 9] have been used to study the
cleavage planes arrangement and their bifurcations. However,
those studies were limited to small tissue samples ~ 60 µm.
Confocal microscopy can offer a very high 3D resolution, but
cannot acquire large image volumes [10, 11]. DT-MRI is also
used to image the myocardial architecture non-destructively;
however, DT-MRI is limited by a low spatial resolution,
comparatively to the myocytes size, to investigate the
microstructure [12].
II. HUMAN HEART SAMPLES
The human heart sample was supplied by the MedicoLegal Institute of Lyon IML HCL (n◦ DC − 2012 − 1588) and
collected during a medical-legal autopsy of a subject who
suffered a violent death. A sample (7 x 7 x 15 mm3) located in
the anterior wall of the left ventricle was extracted from this
heart. The sample preparation procedure and the X-rays phase
contrast imaging (PCI) acquisition technique performed at the
ESRF are described in [13]. A set of 2500 X-rays projections is
acquired and the tissue sample is reconstructed in 3D with an
isotropic spatial resolution of 3.5 × 3.5 × 3.5 μm3.
The sample has a length of 15 mm corresponding to the
thickness of the left ventricle anterior wall from endocardium
to epicardium. The width and thickness of the sample are about
5-6 mm. A sub-volume of this sample is shown in Figure 1.
The cleavage planes are clearly visible (Figure 1) as planar
sections of dark gray color (1). The components of the
circulatory system going through the cleavage planes, such as
blood vessels are also visible (2). Myocytes lying in the
longitudinal section, appear as thin white filaments (3) and in
the transversal section (4) as white quasi circular regions. The
intercellular space corresponds to thin black filaments (5) in
the longitudinal section.
cleavage planes because they are the most important structures
contained in the binary sub-volume. We threshold the FFT to
keep only 4% of the lowest frequency components in order to
select the major orientation. We then perform a Principal
Component Analysis (PCA) on the coordinates of these pixels
as proposed in [14]. This determines the main direction (angles
in 3D) of variation that is orthogonal to the cleavage planes.
Figure 1. 3D reconstructed sub-volume of the left ventricle anterior
sample acquired at the ESRF.
In this subsample of size 3 x 1.8 x 0.3 mm3, we can observe
that the cardiac tissue components are locally aligned in a
principal direction. The cleavage planes appear to be parallel
and the myocytes are packaged between the cleavage planes
and aligned. The myocytes orientation is restricted by the
cleavage planes. The following statistical analysis method is
based on the fact that the cleavage planes are locally parallel
and have a privileged direction.
III. MULTISCALE PROCESSING
We analyze the orientation and spatial organization of the
cleavage planes for three subsamples of the anterior left
ventricle sample: respectively located close to the
endocardium, in the mid-wall and close to the epicardium.
In order to measure the distances between the cleavage
planes, we create a large number of parallel lines crossing the
cleavages planes through the 3D sub-volume (Figure 3). Then
we analyze the binary profiles along these lines (white = 255
for myocyte sheets and black = 0 for cleavage planes). The
sequence length of consecutive 255 values reflects the distance
between the cleavage planes and the sequence length of
consecutive 0 values gives the thickness of the cleavage planes.
The distribution of the length of the two types of sequences,
255 and 0, is obtained, respectively, by computing the two
histograms for the entire set of lines. The statistics about
cleavages plane distances and size are then extracted from
these 2 histograms (Figure 4). The lines are spaced by 3 pixels
(10.5 µm). The total number of lines in each subsample is
about 110 000.
Each 3D reconstructed sub-volume is 7 x 7 x 0.9 mm3 in
size corresponding to 2048 voxels x 2048 voxels x 256 voxels.
Each sub-volume contains 109 voxels representing 4.3 Gb.
Our aim is to extract the cleavage planes structure
automatically. To do so, we develop a multiscale approach.
First, we build a Gaussian pyramid of each sub-volume. Then,
we compute the difference between two levels of the Gaussian
pyramid corresponding to σ = 3 and σ = 44 respectively. This
allows both to suppress highfrequencies corresponding to noise
and slowly varying background while preserving the large
structures. We select the size of the Gaussian filter in
accordance with the size of myocytes we want to eliminate.
The next step is a binarization operation. We apply a 3D Otsu
thresholding [15] globally on the preprocessed sub-volume to
obtain the cleavage planes. The entire process is illustrated in
Figure 2. To investigate the impact of the threshold value on
the cleavage planes detection and their quantification
properties, we vary the threshold on either side of the obtained
threshold. We demonstrate that the evolution of the histogram
corresponding to the distance between the cleavage planes
remains rather low and that the histogram corresponding to the
thickness of the detected cleavage planes remains very similar
when the threshold varies around the “Otsu” Value.
IV. STATISTICAL ANALYSIS OF THE CLEAVAGE PLANES
Once each sub-volume has been binarized, we
automatically detect the preferential average direction of the
cleavage planes using the following strategy: we take the 3D
Fast Fourier Transform (FFT) of a cubic region of interest of
each subsample. The Fourier coefficients of highest amplitude
correspond to the preponderant spatial orientation contained in
the sub-volume. Such an orientation is orthogonal to the
Figure 2. Multiscale processing steps used for extracting the
cleavage planes
The entire process of determining the average orientation,
constructing the series of lines and extracting dimensional
statistics is illustrated in Figure 2 and Figure 3.
corresponding to 185.5 µm. The average distance in the
epicardium is about 39 pixels (136.5 µm). Those results are
presented in terms of histograms in Figure 5. On the other
hand, visually, the orientation of the cleavage planes changes
from endocardium to epicardium. Qualitatively, when passing
from endocardium to mid-wall, the orientation of the cleavage
planes changes by about 90°. Likewise, when passing from
mid-wall to epicardium, the cleavage planes orientation also
changes by approximately 90°. So, the orientations of the
cleavage planes show a range of about 180°.
VI. CONCLUSIONS
In this paper we have presented a multiscale strategy to
automatically extract and analyze some features of the
cleavage planes in a fresh human heart sample. The tissue
sample was imaged in 3D at a very high isotropic resolution
(3.5 µm) using X-rays phase contrast micro CT. We processed
three distinct reconstructed sub-volumes of the sample,
respectively located close to the endocardium, mid-wall and
epicardium. We demonstrate that the proposed method allows
to clearly reveal the cleavage planes in the myocardium and
quantify some statistical properties such as their thickness, the
distances separating them and the evolution of their
orientation through the wall. Those are our first results on the
left ventricle. New cardiac tissue samples are currently being
processed and analyzed.
Figure 3. The. red lines are perpendicular to the main orientation of
the cleavage planes derived from the PCA of the FFT.
V. RESULTS
The above-described method is applied on three sub-regions
of the sample with equal size of 6 x 6 x 0.9 mm3: a region
located at 1.6 mm from the endocardium, a region located at
mid-wall (at a distance of 7.5 mm from the endocardium in the
middle of the sample) and a region located at 2 mm from the
epicardium (i.e. at 13 mm from the endocardium). The
cleavage planes are binarized using the method presented
above. They correspond to the linear black objects in Figure 2
and Figure 3 also including some blood vessels. At the
endocardium, the sample contains trabeculae and papillary
muscles. Figure 4 shows three representative sections of the
myocardial volume, which correspond to the endocardium,
mid-wall and epicardium, respectively. We clearly observe the
presence of cleavage planes represented by blue linear objects.
They are parallel to each other. In the sub-volume close to the
endocardium, the average thickness of the cleavage planes is
10 voxels (35 µm) and the average distance between the
planes is 57 voxels (200 µm). They are longer and less
fragmented in the mid-wall than at the epicardium. The
thicknesses of the cleavage planes in the epicardium and midwall are similar, with an average of about 8 pixels (i.e. 28
µm). In contrast, the distances between consecutive cleavage
planes in the mid-wall and the epicardium are slightly
different; the distances in the sub-volume close to the
epicardium are smaller than in the mid-wall. The average
distance in the mid-wall is approximately 53 voxels
Figure 5. Statistical study of thicknesses (left column) of cleavage
planes and distances (right column) between cleavage planes. The
top, middle and bottom rows correspond to the endocardium, midwall and epicardium sub-regions, respectively.
ACKNOWLEDGMENTS
The authors thank L. Fanton, F. Peyrin, M. Langer, C. Olivier
for their help in obtaining the data. This work was done in the
scope of the LIA Metislab.
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Figure 4. Example of three sections of the myocardium X-Rays
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