Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

Knowing cardiac anatomy is fundamental to understanding cardiac physiology. Studies of functional anatomy have spanned the micro to macro scales, but have largely used animal hearts. Systematic study of donor human hearts rejected for transplantation provides a unique opportunity to develop a comprehensive atlas that spans the microscopic to macroscopic scales.

Through study of the human heart across different scales, we've demonstrated the exquisite innovation of the heart, identified cardiac anatomic structures to improve surgical and interventional approaches and developed resources for anatomic education. The advantage of studying the human heart across different scales is that the detailed study of microscopic structures may be placed in the context of gross anatomy. The use of pressure profusion fixation maintains the physiologically relevant morphology of the heart.

To begin, use a scalpel to dissect 4%paraformaldehyde fixed human heart tissue to fit within the chamber for confocal microscopy. Dehydrate the heart specimens in a graded methanol series for one hour each at room temperature with agitation, followed by overnight immersion in a 66%dichloromethane and 33%methanol solution with agitation. The next day, bleach the sample by performing the indicated steps.

Then rehydrate the specimen with a graded methanol series for one hour each with agitation. After immuno labeling, perform the following steps to prepare the tissue for imaging. Affix a chamber containing adhesive to a slide and apply nail polish around the perimeter of the chamber.

Turn on the upright laser scanning confocal microscope equipped with a 5X lens. Using laser lines appropriate for the emission spectra of the fluorophores used, acquire tile scan and Z-stack images at a resolution of 1024 by 1024. At the micro scale level, tissue clearing of heart samples allows visualization of ganglia containing cardiac neurons and the neural patterning of myocardial innervation.

After turning on a stereo microscope, perform delicate dissections on paraformaldehyde fixed human heart tissue, focusing on structures such as the atrial ventricular node, atrial ventricular node artery and cardiac nerve plexus. Place the heart sample on the scanner bed to acquire a CT image of the heart specimen. Using a 20 to 24 French surgical cannula, canulate the superior vena cava for right heart perfusion, then occlude the inferior vena cava and another pulmonary artery with twine.

Next, perform a progressive dissection of the heart tissue with photographic recordings at each stage of dissection. Using multiple light emitting diode light panels set on C-stands with a wide black dividend background cloth, photograph the heart with a digital single lens reflex camera. Suspend the heart sample on a rotation table to obtain hundreds of multi-directional photographs with a smartphone.

On Scaniverse and Polycam software, generate the 3D model of the heart in FBX format. Suspend the heart sample from a bar across the top of the container. Use a multi detector row CT scanner to conduct CT scans of the heart.

After generating virtual dissection images of the heart, visualize the non-enhanced walls, septa and valves with virtual dissection to produce images similar to gross dissection.