The overall goal of this procedure is to induce cerebral cavernous malformations in neonatal mice, and measure the disease burden using micro-CT. This method can help answer key questions in cerebral vascular disease field, such as cerebral cavernous malformations. The main advantage of this technique is that cerebral cavernous malformation disease burden in neonatal mouse models can be determined accurately and efficiently compared to existing methods.
Visual demonstration of this method is critical, as induction of cerebral cavernous malformation and micro-CT analyzing steps are difficult to learn, because it requires experience and use of sophisticated software. Begin by dissolving 4-HT in 100%ethanol to a concentration of 10 milligrams per milliliter. Prepare 30 microliter alaquads, and then store at minus 80 degrees Celsius.
On the day of use, dilute the alaquad at 4-HT in corn oil to 0.5 milligrams per milliliter. Then use an insulin syringe to introgastricallly inject 50 microliters of 4-HT into P1 neonatal pups to induce experimental CCM lesions. Begin sample preparation by filling a 10 milliliter syringe equipped with a 29 gauge needle with three milliliters of 2%paraformaldehyde and PBS.
Then perform intracardiac profusion on euthenized 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 and PBS. Post fix the brains overnight at 4-HT 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 hind brain on an aluminum holder in the micro-CT system.
Set the scanning parameters. Use 540 projections and a two second exposure time with source conditions of 50 kilovolts and 10 Watts to acquire the tomographic datasets. Radio graphs from the scan are reconstructed automatically by hardware-based projection reconstruction software, supplied by the micro-CT system, producing an image series of 16-bit axial slices.
Lugol's iodine contrasted micro-CT sufficiently detected CCM lesions. Scanned X-ray images were reconstructed to produced 3D images of the mouse brain, which allowed visualization of entire lesions in the brain parenchyma, at different depths and orientations, and assessment of the structure in 3D location of lesions in the brain. This table shows an example of quantitative output of the labeled individual lesions.
After its development, this technique paved way for researchers in the field of vascular disease to explore mechanism and therapeutic options for cerebral cavernous malformations in mouse models. After watching this video, you should have a good understanding of how to induce CCM lesion in mouse models, and how to acquire and analyze CCM lesion burden with micro-CT technique.
