Extended confocal microscopy of myocardial laminae and collagen network

Abstract

Key words. Confocal fluorescence microscopy, extended volume image, myocardial blood vessels, myocardial collagen, myocyte organization, picrosirius red, threedimensional cardiac microstructure. SummaryVentricular myocardium has a complex three-dimensional structure which has previously been inferred from twodimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusionstained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 mm in a contiguous mosaic across the surface. Approximately 50 mm was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 × 800 × 800 mm 3 spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart.

Effects of Angiotensin II Type 1 Receptor Antagonist on Smooth Muscle Cell Phenotype in Intramyocardial Arteries from Spontaneously Hypertensive Rats

“…After the hemodynamic measurement, the rats were euthanized with a sodium pentobarbital overdose, and the hearts were excised and weighed. Some of the excised hearts were perfused and fixed with heparinized saline (20 units/ml) followed by Bouin's solution via retrograde infusion into the ascending aorta at a pressure of 90 mmHg (14), and then paraffin-embedded 4-µm slices were stained with Sirius red for histological analysis. Other heart tissue sections were snap-frozen with Optimal Cutting Temperature (O.C.T.)…”

Reconstruction and Quantification of Diffusion Tensor Imaging-Derived Cardiac Fibre and Sheet Structure in Ventricular Regions used in Studies of Excitation Propagation

“…When data from seven hearts were combined, a bimodal distribution of angles was found with two distinct orientations of the cardiac laminar structure lying on average at 45.5 • and 117.6 • [29]. These findings are backed by histological studies [1,2,11,16,19,27,67] that have also identified two distinct populations of sheets in combined data that correlate well with the DTI measurements. Our measured sheet angles for lateral sectors show large intra-and interheart variability, particularly in basal regions (see Figs.…”

“…The data presented here also offer some possible explanations for the differences in the histologically-derived descriptions of sheet morphology in the literature. Some reports [11,46] describe a single population of sheets that bend through the cardiac wall, while others describe dual populations [1,2,11,16,19,27,67]. Gross variation in sheet structure between individual hearts will result in differing reports, as will local variation in sheet orientation associated with the precise positioning of the regions selected for characterisation.…”

Cardiovascular Mechanics

“…1.4 (b). The orientation of the sheets vary both transmurally and in the apico-basal direction [24,25,27,28]. Labeling the local myocyte direction as the fiber direction, we may thus characterize the myocardium as an orthotropic material with a fiber, sheet and sheet-normal direction labeled f, s and n, respectively.…”

“…The myocytes in the myocardium of the left ventricle of the heart is in general organized in a right-handed helical pathway from the endocoardium towards the midwall, and a left-handed helical pathway from the midwall towards the epicardium, [22][23][24]. Furthermore, the myocytes are bundled and form layers with a direction that vary through the thickness of the ventricle wall, [24,25,27,28]. This organization of the myocyte, in both a fiber and sheet direction, is responsible for the twisting motion of the heart during systole [127].…”