Fig 1.
Tissue clearing process of the hearts.
Bright-field images of heart samples during the clearing procedure: (A) After the phosphate-buffered saline wash, (B) after fixation with 4% paraformaldehyde, (C) day 1 of the CUBIC-1 process, (D) day 14 of the CUBIC-1 process, (E) after immunostaining, and (F) after the CUBIC-2 process. Heart samples are made transparent through the CUBIC-1 process (A-D). After immunostaining, samples have a decreased transparency, but the CUBIC-2 treatment restores the samples to a high transparency. Images are acquired using a stereomicroscope. The scale bars represent 2 mm.
Fig 2.
Three-dimensional (3D) distribution of sympathetic nerves and coronary vessels in the heart.
(A) X-Y plane image of tyrosine hydroxylase (TH, green)-stained heart sample. Sufficient transparency is achieved, and the transmural distribution of sympathetic nerves is visualized. (B) 3D images of TH (green)-stained and α-smooth muscle actin (red)-stained heart samples. (C) A higher magnified view of the boxed region in B. Cardiac nerves distributed along the epicardial surface with nerve branches penetrating into the myocardium and wrapping around coronary vessels. Images are obtained using a light-sheet microscopy (Lightsheet Z.1, Carl Zeiss). Scale bars represent 500 μm.
Fig 3.
Spatial relationship between sympathetic nerves and coronary vessels.
X-Y plane (A) and three-dimensional (B) images of tyrosine hydroxylase (green)-stained and α-smooth muscle actin (red)-stained heart samples. Images demonstrate that nerve fibers are distributed around coronary vessels. Images are obtained using a confocal microscope (LSM 700, Carl Zeiss). Scale bars represent 200 μm. (C) High resolution images of sympathetic nervous distribution around the coronary vessels. (D) Higher magnified view of the boxed region in C. Numerous hairline nerve branches are allocated around the coronary vessels. Images are acquired using a confocal microscope (LSM 880, Carl Zeiss) with Airyscan (Carl Zeiss). Scale bars represent 50 μm (C) and 20 μm (D).
Fig 4.
Three-dimensional (3D) imaging of sympathetic nerves and coronary vessels in the post-myocardial infarction (MI) mouse heart.
(A) Three-D image of the heart 2 weeks after inducing MI. Heart samples are immunostained with tyrosine hydroxylase (green) and α-smooth muscle actin (red). Sympathetic nerves are abruptly extinguished at the site of the ligation. Arrowheads indicate the left anterior descending coronary artery. The dashed line indicates the site of ligation. The right panel shows a higher magnified view of the boxed region in the left panel. Images are obtained with a light-sheet microscope (Lightsheet Z.1, Carl Zeiss). Scale bars represent 500 μm. (B) Three-D image of the ischemic border zone in the post-MI heart. The right panel shows a higher magnified view of the boxed region in the left panel. In the ischemic border zone proximally adjacent to the site of ligation, numerous fine nerve fibers are distributed in a disordered manner. Images are obtained with a confocal microscope (LSM 700, Carl Zeiss). Scale bars represent 200 μm (left panel) and 100 μm (right panel). (C) Analyses of sympathetic nerves in post-MI mice. The nerve volume and surface area are significantly increased in the ischemic border zone and decreased in the infarct area of post-MI hearts compared to intact mice hearts (n = 5, *p<0.05, by Steel-Dwass test). Error bars represent standard error of the mean. IBZ, ischemic border zone.