Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

This protocol provides micro structural insight into structural pathological diseases at the whole organ scale using microcomputer tomography. In this protocol, a specialized tissue preparation method is implemented to optimize high resolution imaging of whole heart samples from translational large mammalia models in humans with selective contrast enhancement of structural diseases. The cardiac microstructure such as the distribution of fibrosis and the organization of the excitable myocardium underlies a broad spectrum of cardiac diseases and sets the scene for the life threatening arrhythmia.

Demonstrating the procedure will be Nestor Pallares-Lupon, a postdoc and PhD graduate for my laboratory. To begin, prepare a cardioplegic solution containing sodium heparin and store the solution at four degrees Celsius. Next, prepare PBS/EDTA by first adding EDTA to one liter of distilled water for a 10 millimolar final concentration.

Then increase and maintain the pH of the solution at 12 using sodium hydroxide to dissolve the EDTA. Once the EDTA is fully dissolved, lower the pH to 7.4 using hydrochloric acid. Then add one foil pouch of PBS to obtain a 0.01 molar solution with pH 7.4.

Store the prepared solution at room temperature. Finally, prepare a 1%ethanol PMA contrast agent solution by adding 10 grams of PMA to one liter of absolute ethanol. Store the prepared solution at room temperature.

Prepare a one liter reservoir supported 80 centimeters above a dissection dish, and couple a thermoplastic tube to a drain port of the reservoir. Fix a three-way tap to the drainage tubing and couple further thermoplastic tubing to each free port on the three-way tap. Fix two-way taps to the free ends of the tubing.

Next, fill the reservoir with the cardioplegic solution supplemented with heparin. Open the taps to allow the cardioplegic solution to drain removing all air bubbles. Then close the two-way taps.

After extracting the heart from a euthanized animal, place it in cold cardioplegic solution stored on ice until ready for dissection. Then prepare cannula for the left and right coronary osteo by cutting five centimeters of PTFE tubing and heating one end by placing the tip next to a naked flame. Once one millimeter of the tip begins to melt and becomes translucent, press the tip against a hard heat resistant surface to shape a ridge at the cannula tip to prevent the cannula from slipping out of the vessels, then insert one centimeter of the non-heated end of each cannula into the two ends of the drain reservoir drainage tubing.

Next, remove the aortic cannula and localize the left and right osteo of the coronary arteries under the cold cardioplegic solution. Using pointed scissors, carefully separate the aortic root from the surrounding tissue above and below the coronary osteo to enable threading of a zero gauge silk suture under the coronary vessel. Then open the two-way taps and insert the cannula tips into the coronary osteo.

With the cannula tips extending one to two centimeters into the osteo and beyond the suture placement, tie off the cannula. Next, rinse the heart while gently massaging the ventricles for 15 minutes until the heart is cleared of blood. After rinsing, close the two-way taps, disconnect them from the three-way tap and transfer the heart to a one liter plastic chemical resistant container containing 500 milliliters of PBS/EDTA solution.

Recirculate the PBS/EDTA solution in the thermoplastic tubing under a fume hood using a peristaltic pump with two channels. Prime the pump tubing until the tubing is absent of air bubbles. Then peruse each coronary artery cannula by recirculation at room temperature for two hours at 80 milliliters per minute.

After ensuring that the fume hood is operational, stop the pump, drain the solution from the container and replace it with 10%formalin for fixation for one hour at 80 milliliters per minute. Peruse the heart using a series of incrementing ethanol concentrations, starting with 20%ethanol for a minimum of three hours. Then replace the 20%ethanol with 30%ethanol and perfuse for two hours.

Continue the perfusion by incrementing the ethanol concentration at each iteration with minimum perfusion of one hour at each concentration. To reinforce the heart tissue before air drying, recirculate an equal mix of ethanol and HMDS for 10 minutes followed by 100%HMDS for a further two hours. Stop the perfusion pump, then disconnect the cannula from the tubing and suspend the heart from an aortic suture inside the fume hood.

Carefully slide a zip lock bag over the heart and close the bag seal over the suture to reduce exposure of the heart to the circulating air. Allow the heart to dry through evaporation for one week. For diffusion loading contrast agents, remove the bag.

Wash the heart in 100%ethanol for 15 minutes with agitation before immersing it in 100%ethanol supplemented with 1%PMA for 48 hours under vacuum, then cover the heart with a zip lock bag as demonstrated earlier and allow it to dry. Mount the air dried heart onto an appropriate sample holder. To prevent any movement during the x-ray micro CT measurements, use a clamp anchored to the sample holder and secure the heart via the dried and rigid aorta.

Mount the sample holder into the micro CT scanner. Next, open the software and initiate the x-ray micro CT system. Then set the x-ray filter aluminum to one millimeter.

X-ray source voltage to 60 kilovolts and current to 120 microamperes. Additionally set image dimensions to 2, 016 by 1, 344 pixels and pixel size to 20 micrometers. Scout x-ray transmission images along the length of the support to determine the overall imaging field in the heart's longitudinal access.

For scanning, use a rotation step of 0.18 degrees, a frame averaging of five and a sample rotation of 180 degrees by deselecting the option for a 360 degree acquisition. Select the offset scanning mode to image the entire width of the sample support. After scanning, use the software for tomographic reconstruction of an isotropic three-dimensional image volume.

In the end recon software, use acquisition related artifact correction, including beam hardening effects of 10%and ring artifact reduction of eight. Next, using Data Viewer software, visualize the reconstructed data stack. Digitally orientate the sample within the image boundaries to realign the samples long and short axis with the three principle a axis of the image volume.

Finally, crop the image volume in all three axis to remove outer background layers of the image and maximally reduce the total image size. X-ray transmission imaging of air dried pig hearts shows major anatomical landmarks including the ventricular cavities and the muscle wall. Tomographic reconstructions of three dimensional image volumes showed distinct separation between tissue and background at epicardial and endocardial boundaries.

Mason's trichrome staining showed collagen positive staining at the epithelial and endothelial layers paravascularly in the sub-epicardial tissue. Bright field illumination showed darker coloration in collagenous structures after PMA staining, supporting the preferential accumulation of PMA. PMA stained fluorescent images of ventricular tissue sections had PMA induced loss of fluorescence at sites of collagen.

Where a heart sample stained with a contrast agent via perfusion prior to air drying, image reconstruction revealed highly patchy staining within the myocardial compartment. Moreover, low contrast tissue showed poor separation from the background intensity. Micro CT imaging of a sheep part suffering from chronic myocardial infarction revealed a central dense fibrotic lesion surrounded by a loose and heterogeneous border zone.

The greatest signal intensities of reconstructed image volumes at the tissue boundaries and scar regions are shown here. Contrast agents poorly stained the healthy myocardium, yet micro structural contrast was retained. At the border zone, scar tissue was interspersed with surviving myocardium.

Dense fibrosis appeared transmural yet textured indicating variances in composition. Transmural left ventricular region of PMA stained tissue preparation showed selective staining for collagen and no staining in regions of surviving myocardium. Longer perfusion times using ethanol are recommended to ensure the complete exchange of the heart's water content with ethanol.

Interactions between HMDS and the water produce heat, which may damage the sample. A strong advantage of this procedure is the ability to validate micro CT imaging using histology. Air dried samples can be directly embedded in paraffin for sectioning staining, and microscoping imaging.