Three-dimensional histology: tools and application to quantitative assessment of cell-type distribution in rabbit heart

Abstract

Aims: Cardiac histo-anatomical organization is a major determinant of function. Changes in tissue structure are a relevant factor in normal and disease development, and form targets of therapeutic interventions. The purpose of this study was to test tools aimed to allow quantitative assessment of cell-type distribution from large histology and magnetic resonance imaging- (MRI) based datasets.

Methods and results: Rabbit heart fixation during cardioplegic arrest and MRI were followed by serial sectioning of the whole heart and light-microscopic imaging of trichrome-stained tissue. Segmentation techniques developed specifically for this project were applied to segment myocardial tissue in the MRI and histology datasets. In addition, histology slices were segmented into myocytes, connective tissue, and undefined. A bounding surface, containing the whole heart, was established for both MRI and histology. Volumes contained in the bounding surface (called 'anatomical volume'), as well as that identified as containing any of the above tissue categories (called 'morphological volume'), were calculated. The anatomical volume was 7.8 cm(3) in MRI, and this reduced to 4.9 cm(3) after histological processing, representing an 'anatomical' shrinkage by 37.2%. The morphological volume decreased by 48% between MRI and histology, highlighting the presence of additional tissue-level shrinkage (e.g. an increase in interstitial cleft space). The ratio of pixels classified as containing myocytes to pixels identified as non-myocytes was roughly 6:1 (61.6 vs. 9.8%; the remaining fraction of 28.6% was 'undefined').

Conclusion: Qualitative and quantitative differentiation between myocytes and connective tissue, using state-of-the-art high-resolution serial histology techniques, allows identification of cell-type distribution in whole-heart datasets. Comparison with MRI illustrates a pronounced reduction in anatomical and morphological volumes during histology processing.

Keywords: Cardiac MRI; Computational models; Connective tissue; Myocytes; Serial histology.

Figure 1

MRI-based tissue segmentation. (A–C) Down-sampled MRI image in three orthogonal planes. (D, E) Corresponding boundary surface segmentations for anatomical volume determination. (F, G) Detailed tissue segmentations to separate tissue from non-tissue (including clefts, interstitial spaces, blood vessels) for morphological volume calculation. (H) 3D iso-surface visualization of bounding surface segmentation obtained from MRI. LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; Ao, aorta.

Figure 2

Histology-based tissue segmentation. (A–C) Histology slices #460 containing the RV cavity), #925 containing LV cavity and part of the septum, and #1387 through the LV cavity (out of 1850 sections in the whole-heart histology stack). (D, F) Corresponding tissue segmentation. Note that the black marker lines at the top left corner in (A–C) have been masked out in the segmentation. (G, H) Volumes measured in slices with valid segmentations (blue curve), and after interpolation to correct for missing sections (red curve). (G) Bounding contour segmentation yielding anatomical volume. (H) Tissue content segmentation yielding morphological volume. LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium.

Figure 3

Histology-based cell-type segmentation. (A) Trichrome-stained rabbit heart section #925, containing. Ao, aorta; LV, left ventricle; MV, mitral valve; scale bar 3.5 mm. Rectangles i, ii, and iii correspond to locations of tissue type segmentation in panel (D). (B, C) Two sample histology images that are shown as trichrome-stained sections (B-i and C-i); after myocyte segmentation (B-ii, C-ii), and after connective tissue segmentation (B-iii, C-iii). (D) Enlarged aspects of the trichrome-stained section in panel (A). Image area dominated by myocytes (D-i), connective tissue (D-ii), or cells that are ‘undefined’ by the algorithm (D-iii). Arrows point to location of sample pixels analysed. (E) Summary data showing the ratio of myocyte: connective tissue pixels in the entire stack of slices, ordered in the same sequence as in Figure 2G and H (red: myocyte pixels, blue: connective tissue pixels). White lines: missing sections in the histology stack.