Rapid Whole-Mount High-Resolution Imaging of Small Animal Vasculature for Quantitative Studies

We work to understand the mechanisms of disease formation, particularly surrounding congenital heart defects and whether changes in hemodynamic flow contribute to or compound disease formation. Nano-computed tomography, small animal magnetic resonance imaging, 4D ultrasound, and optical coherence demography are all used to gather high resolution, quantitative imaging scans for computational modeling.

Our protocol addresses the need for a rapid and readily accessible quantitative vascular imaging technique that can be applied to a variety of animal models, including their embryos and organs. Our protocol is time-efficient, taking sample prep time from four weeks to just three days. It uses minimal hazardous clearing compounds, and it does not rely on vascular markers that are not present in early embryos.

Now that we've established a technique to routinely gather high resolution, subject-specific volumetric scans, we can focus on creating new disease models resulting from genetic and mechanical perturbations.

[Narrator] To begin, position the avian embryo, ranging from Hamburger Hamilton Stage 18 to Stage 34 under a stereoscope or on a dissection table. Cut around the embryo from the egg yolk using curved tip scissors, and then using a transfer pipette or a spatula, transfer the embryo into a 35 millimeter Petri dish filled with warm Tyrode's solution. Under the stereoscope, using fine tip forceps, make approximately 0.1 millimeter incisions in the membranes to remove the chorion and Lantus covering the embryo. Gently pull away the membranes, along with the pericardial membrane surrounding the heart. Transfer the embryo into a new clean Petri dish filled with warm Tyrode's solution to maintain heart activity. Now, fill a five milliliter plastic syringe with warm Tyrode's solution. Trim the wide end of a to 20 microliter plastic pipette tip and securely attach it to the flat end of the syringe. To construct an injection line, attach a segment of 0.03 inch inner diameter silicone tubing to the pipette tip syringe assembly, followed by a glass capillary microneedle to the opposite end of the tubing. Then mount the microneedle onto the micro injection holder, connected to a micro-manipulator, and purge all air bubbles from the silicone tubing and microneedle. Using the micro manipulator, insert the microneedle into the apex of the heart and slowly inject Tyrode's solution to perfuse the embryo. Next, use the micro-manipulator to retract the injection line and reorient the microneedle so that it remains stable during further preparations. Then detach the silicone tubing from both the needle and syringe. Prepare for endo painting to label the vascular endothelium with green fluorescence. Using a micro pipette, load 20 to 40 microliters of FITC Poly-L-Lysine stock solution into the silicone tubing. Reattach the Tyrode's solution-filled syringe, and using the micro manipulator, slowly inject the solution into the heart apex, reinserting the needle if necessary. Then remove the needle after FITC Poly-L-Lysine is diffused, ensuring no air bubbles enter the heart. Let the FITC Poly-L-Lysine remain in the embryo for 5 to 10 minutes. Then fill a five milliliter syringe with 4% paraformaldehyde and attach it to the silicone injection tubing to prepare for a third injection step. Perfuse the embryo with 4% paraformaldehyde until the cardiovascular structures are completely filled and the tissue becomes more opaque. Gently place the embryo into a two to five milliliter vial, filled with enough 4% paraformaldehyde to submerge the sample and incubate it overnight at four degrees Celsius in a shaker. Carefully pipette out the 4% paraform aldehyde from the vial. Add fresh PBS to the embryo and incubate at room temperature for 30 minutes while gently shaking to wash the sample. To dehydrate the embryo, place it under a fume hood and carefully pipette out the PBS. Incubate the embryo sequentially in a graded series of methanol concentrations for one hour each at room temperature. Then incubate the embryo in fresh 100% methanol overnight at room temperature. To remove lipids, add a two to one volume to volume dichloromethane to methanol solution to the embryo. And incubate for three hours with gentle shaking at room temperature. Remove ethyl cinnamate from refrigeration at four degrees Celsius and allow it to thaw to room temperature. Wash the embryo in fresh 100% dichloromethane at room temperature for 15 minutes while shaking. Remove dichloromethane using a pipette and incubate the embryo in 100% ethyl cinnamate until the embryo clears, which takes approximately one hour. Mount the glass capillary onto the sample holder and fill the imaging chamber with ethyl cinnamate. Now, gently lower the embryo into the chamber. Adjust imaging parameters according to specifications and perform light sheet imaging. Create a new SV project under File, and upload a high resolution light sheet imaging stack after right clicking on Images and selecting Add or Replace image. To create a path line, right click on Paths and select Create Path. Place path markers along a vessel of interest, and repeat this process for each vessel to be analyzed. For each path line, right click on Segmentations and select Create Contour Group to begin segmentation. Select the vessel path and double click on the vessel name under Segmentation to initiate manual segmentation of the 2D cross sections. After segmenting all vessels, right click on Models and choose Create Model. Finally, select PolyData type, enter a model name, click Create Solid Model, and select all relevant segmentations. Using the optimized imaging protocol, clearly outlined vessel lumens were obtained across different embryonic stages, revealing pharyngeal arch arteries, dorsal aorta, and cardiac structures such as ventricles and atria in green fluorescence, Light sheet fluorescence microscopy enable detailed 3D reconstruction of the outflow tract, pharyngeal arch arteries, and dorsal aorta, allowing cross-sectional and depth-resolved views of vascular anatomy. Optical clearing of the embryos significantly improved the visibility of internal vasculature under fluorescence, particularly in the GFP channel where the labeled vessels became more distinct post clearing. Anatomical reconstructions and computational simulations of the day five chick embryo pharyngeal arch arteries revealed distinct blood flow paths to cranial and caudal regions and showed peak wall shear stress values aligned with prior nano-computed tomography studies.