Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

1. Clearing Blood from Vasculature

1. Anesthetize mouse by intraperitoneal injection of 100 mg/kg ketamine with 10 mg/kg xylazine diluted in 0.25 ml saline.
2. Once anesthesia has been confirmed by no response to a paw pinch, use scissors to make an incision across the chest with scissors just below the xyphoid process, cut through diaphragm, and cut up between dorsal and ventral segment of ribcage to expose thoracic cavity. Expose heart by folding up sternum and adjacent chest wall and secure with hemostat. Open the pericardial sac with tweezers to expose the heart.
3. Turn on perfusion pump (100mmHg pressure) connected to warm PBS plus heparin (5 unit/ml) solution in hot water bath at 37°C and tubing with a blunt 22 gauge needle on the outflow.
4. Insert the needle into the left ventricle, near the apex of the heart. Immediately cut open the right atrium to enable systemic blood outflow and continue flushing until blood is removed from circulation.

2. Perfusion of Casting Agent into the Vascular System

1. Prepare Microfil (Flow Tech, Inc. Carver, MA) casting solution per manufacturer’s protocol: Mix 5 ml of MV diluent with 4 ml of filtered MV compound. Add 450μl (5%) of catalyst (MV curing agent). Solution working time is 20 minutes, but mix immediately before use to ensure proper viscosity.
2. Withdraw casting agent solution into a 10ml syringe, attach a blunt 20 gauge needle to the syringe and fill needle with solution.
3. Insert needle into the left ventricle through the same hole used for flushing the blood. Secure needle into place with hemostat clamp on the heart.
4. Inject casting agent solution slowly into the left ventricle at approximately 3 ml/min. Excess pressure may lead to incorrect flow of casting agent or potential vessel rupture.
5. Look for signs of successful perfusion, including: visualization of casting agent in intestine and liver vasculature, distal limb discoloration, and nose and tongue discoloration. Readjust needle if necessary to achieve proper perfusion.

3. Collection and Processing of Tissue

1. Remove brain and place in 4% paraformaldehyde overnight at 4°C.
2. Dehydrate brain by placing from paraformaldehyde directly into increasingly concentrated EtOH at room temperature: 25% EtOH, 1 day; 50% EtOH, 1 day; 75% EtOH, 1 day; 95% EtOH, 2 days; and 100% EtOH, 2 days.
3. Clarify brain tissue around blood vessels by immersing the perfused brain in methyl salicylate for 2 days at room temperature.
4. Vasculature of the whole unsectioned clarified brain can be imaged with brain immersed in methyl salicylate under a dissecting microscope (Fig. 1) (Leica MZFL III microscope, Leica Microsystems, Bannockburn, IL) or brightfield microscope (Fig. 2) (Leica DM/LS, Leica Microsystems).
5. Reclarification can be done if tissue is not completely cleared. From methyl salicylate, transfer brain to 95% EtOH for 2 days, 100% EtOH for 2 days, and methyl salicylate for 2 days.

4. Micro-CT Imaging or Histological Application- Tissue Preparation

1. The brain can be imaged using micro-CT directly after clarification or after re-hydration (Fig. 4).
2. To prepare brain tissue for histological analysis, rehydrate the brain through serial diluted EtOH: 100% EtOH, 1 day; 95% EtOH, 1 day; 75% EtOH, 1 day; 50% EtOH, 2 days; 25% EtOH, 2 days; and store in PBS. The brain can then be embedded in paraffin using a standard procedure.

5. Representative Results

Following vascular casting, the cerebrovasculature can be seen macroscopically on the brain surface (Fig.1A). The vascular tree inside the parenchyma can be visualized following clarification (Fig. 1B). The detailed vascular structure can be imaged and analyzed under a brightfield microscope (Fig. 2), dissecting scope (Fig. 3), and with micro-CT scanning (Fig. 4). Using brightfield microscopy, the vasculature can be viewed at different focal planes without sectioning the brain (Fig. 2A) as well as individual microvessels at higher magnification (Fig. 2B). Under a dissecting scope, one can see the entire vascular structure in one focal plane, allowing better visualization of the three dimensional structure of the cerebrovasculature (Fig. 3). Images taken under a dissecting microscope can show large vessel morphology and whole brain vasculature, but lack the details shown in pictures taken with brightfield microscopy (Fig. 2B). This technique has allowed us to detect irregular vessels in our experimental BAVM adult mouse model². Figure 5 shows an example of the same irregular vessels detected by micro-CT scanning (Fig. 5A) and brightfield microscope (Fig. 5B). Together, this data shows that we can analyze the same sample with multiple imaging tools after vascular casting.

Figure 1. Whole mouse brain following vascular casting. Before (A) and after (B) clarification by methyl salicylate solvent. Vessels can be clearly visualized on the brain surface (A) and in parenchyma following clarification procedure (B).

Figure 2. Brightfield microscope images of clarified vascular casted brain. Brain can be imaged at various planes and at different magnification levels. Scale Bar 200μm (A) and 50μm (B).

Figure 3. Dissecting microscope image at 1x showing whole adult mouse brain cerebrovasculature from a dorsal view.

Figure 4. Micro-CT scanning image of the same whole adult mouse brain shown in Figure 3 from a dorsal view.

Figure 5. Application of vascular casting method to detect a brain arteriovenous malformation. A) Micro-CT scan of vascular casted brain shows region of enlarged, irregular AVM-like vessels in right hemisphere from a ventral view. Enlarged image of region (white box) is shown on the right. B) Same irregular vessels visualized under brightfield microscope at 50x magnification. Scale bar 50 μm.