This article details a method for imaging cerebral cavernous malformations (CCMs) in mouse pup brains using micro-CT. The process involves chemical fixation, staining with iodine, and advanced imaging techniques to visualize abnormal blood vessel formations.
Take a chemically-fixed mouse pup brain with cerebral cavernous malformations or CCMs —clusters of abnormal blood vessels filled with stagnant blood.
Isolate the hindbrain, rinse with buffer, and incubate in an iodine-based staining solution.
Iodine penetrates the permeable blood vessels in CCM areas, binds to blood components, and enhances lesion contrast.
Air-dry the tissue to remove the excess staining solution.
Place the tissue in a tube and seal to prevent tissue shrinkage.
Secure the tube inside a larger tube with sponges and mount it vertically in the micro-CT holder.
Initiate the scan.
The X-ray source emits concentrated X-rays that pass through the tissue.
Iodine-stained areas absorb more X-rays than the surrounding tissue.
The detector records the transmitted X-rays, generating images where the CCMs appear brighter.
The system rotates the tissue, capturing 2D images from multiple angles.
Specialized software reconstructs these images into a 3D model, revealing CCM locations in the brain.
Begin sample preparation by filling a 10-milliliter syringe equipped with a 29-gauge needle with three milliliters of 2% paraformaldehyde in PBS. Then perform intracardiac perfusion on a euthanised P8 pup, by inserting the needle into the ventricle of the mouse heart, and slowly injecting the full volume of paraformaldehyde.
After using scissors to detach the head, remove the skin from the head and peel off the skull, using forceps to dissect the whole brain into 4% paraformaldehyde in PBS. Postfix the brains overnight at 4 degrees Celsius. The following day, detach the hind brains using forceps and rinse with PBS solution.
Then, incubate the hind brains in Lugol's iodine solution for 48 hours. Following the incubation, briefly air-dry the hind brains to remove excess Lugol's iodine solution. Then, pack the Lugol's iodine-stained hind brains in 0.65 microliter microcentrifuge tubes, and seal completely with plastic paraffin film to avoid tissue shrinkage. Place the microcentrifuge tubes in five-milliliter plastic tubes with sponges to prevent them from moving during the scan.
Vertically mount the tube containing a hindbrain on an aluminum holder in the micro-CT system. Set the scanning parameters. Use 540 projections in a two-second exposure time, with source conditions of 50 kilovolts and 10 watts to acquire the tomographic data sets. Radiographs from the scanner reconstruct it automatically by hardware-based projection reconstruction software supplied by the micro-CT system, producing an image series of 16-bit axial slices.
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