Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

This novel tissue clearing protocol improves existing technology to allow for a more accurate in vivo look at specific cell types in the heart and their behavior under injury conditions. This protocol is quick and fairly simple. It allows for the maintenance of marca fluorescence with minimal interference from background tissue auto fluorescence without requiring cell or tissue staining.

Begin by using a 70%ethanol soaked gauze pad to clean the ventral surface of the experimental mouse. Use surgical scissors to make a three centimeter transverse incision, approximately three meters below the xiphoid process and deglove the mouse from the abdomen to the xiphoid process to separate the skin from the underlying abdominal wall tissue. Make a two centimeter transverse incision in the subcutaneous abdominal wall tissue, three centimeters below the xiphoid process and make a vertical cut from this incision up the midline through the rib cage.

Pin the rib cage back to expose the heart. And use a 10-milliliter syringe equipped with a 27 gauge needle to inject cold PBS into the superior vena cava and aorta. When all of the blood has been flushed from the heart, deliver 10 milliliters of cold 4%PFA into the superior vena cava and aorta to begin the fixation process.

When all of the fixative has been flashed, excise the heart and use a straight blade scalpel to separate the atria, right ventricle and septum and left ventricle. Place the heart into a 15-milliliter conical tube of cold 4%PFA for an overnight incubation at four degrees celsius on a nutator. The next morning, add a 0.25%photo initiator solution to a freshly prepared hydrogels solution and transfer the heart to the hydro gel.

Wrap the conical tube in foil for an overnight incubation at four degrees celsius without physical disturbance. The next morning warm the tube in a 37 degrees celsius bead bath for 2.5 hours before using forceps to carefully transfer the heart into a 15-milliliter tube of PBS. Wash the heart three times in PBS for one hour per wash at 37 degrees celsius on a nutator and transfer the heart into the basket of an active electrophoresis machine.

Secure the lid in place and fill the active electrophoresis chamber reservoir with electrophoresis clearing solution. Once the chamber is full, submerge the basket in the solution and tighten the cap on the electrophoresis machine into place. Then run the electrophoresis machine at 1.5 amps and 37 degrees celsius for 1.5 hours.

After electrophoresis, check the heart's visually to ensure that no opaque tissue remains. Wash the cleared heart three times in a 15-milliliter two for one hour in fresh PBS at 37 degrees celsius per wash on a nutator. Before submerging the heart in decolorizing agent to reduce heme auto fluorescence.

After 48 hours, wash the heart three times in PBS as demonstrated and equilibrate the heart in freshly prepared RIMS for 48 hours prior to imaging. For 3D imaging of the cleared heart, first, place a thin layer of vacuum grease onto the bottom of a 3D printed bottom reservoir, the same height as the sample being imaged. Fill the reservoir with RIMS and use a pipette tip to remove any bubbles.

Carefully place the heart in the RIMS with the left ventricular wall facing up, taking care of that no bubbles are introduced into the solution. Use vacuum grease to attach a glass cover slip to the bottom surface of a 3D printed top reservoir piece and place the top reservoir coverslip down onto the bottom reservoirs, taking care to avoid bubbles. Then fill the top reservoirs with glycerol.

Use a confocal microscope to image the cleared hearts. Combining refined cardiac tissue clearing with 3D imaging allows an advanced detailed visualization of cardiac fibroblasts. Using this imaging technique, fibroblasts are observed to be densely packed and to have a spindled morphology in uninjured hearts.

After ischemia reperfusion injury, a loss of cardiac fibroblasts is seen in the ischemic region for the first three days after injury. By day seven and 14, fibroblasts have migrated or proliferated to repopulate the injured tissue area. By day 28, the cardiac fibroblast population surrounding the injured areas of the heart are at their greatest density.

One and a half days after myocardial infarction injury, few fibroblasts remain within the injured left ventricle. By day three however, the fibroblasts expand and are present in most of the left ventricle. In contrast to ischemic surgery, infusion of angiotensin two and phenolefrin over several weeks does not result in cardiac tissue loss and wall thinning.

Instead, the fibroblast align along the axis of the right handed helix contraction pattern as seen when using the refined to tissue clearing protocol. In addition, after angiotensin two and phenolefrin treatment, the fibroblasts appear small and rounded as opposed to the spindle shape observed in models of ischemia reprofusion and myocardial infarction injury. The electrophoretic clearing needs to be performed carefully especially when working with hearts that have undergone injury protocols.

This protocol can be used to assess how cardiac fibroblasts react to injury in vivo. Different fluorescent markers can also be used to understand how the heart responds to injury at the cellular level.