The overall goal of this procedure is to visualize the cerebral vasculature of the adult mouse in 3D. This is accomplished by first opening the thoracic cavity and clearing all the blood from the vasculature through intraoral perfusion of PBS With heparin following perfusion, casting agent is injected into the left ventricle to perfuse the entire circulation system. The perfused brain is then removed and fixed in Paraform aldehyde.
The final step of the procedure is to dehydrate and clarify the brain tissue with ethanol and methyl salicylate. Ultimately, results can be obtained that show a transparent mouse brain whose vascular tree can be imaged under a bright field or dissecting microscope, and by micro CT scan. Hi, my name's Spen Walker at the University of California San Francisco.
Today we're going to be making a vascular cast of the adult mouse brain. The main advantages of this technique over existing methods such as angiography, are that it can be applied to small animals such as the mouse. There's no need for radioactive material, and it is a relatively simple procedure to perform.
It has brought adaptation to various analytical tools, including brightfield, microscopic analysis, CT scanning, due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. Okay, let's get started. Anesthetize the mouse by intraperitoneal injection of ketamine and xylazine diluted in saline and confirmed full anesthesia by paw pinch.
Once anesthesia has been confirmed, use scissors to make an incision across the chest just below the xiphoid process. Next, cut through the diaphragm and then up between the dorsal and ventral segment of ribcage. To expose the thoracic cavity, fold up the sternum and adjacent chest wall and secure them using a hemostat to expose the pericardial sac.
Then open the pericardial sac with tweezers if necessary to expose the heart With the perfusion pump set at 100 millimeters of mercury, begin perfusing PBS plus five units per milliliter of heparin warm to 37 degrees Celsius. Insert the blunt 22 gauge needle connected to the pump outflow 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 the circulation. First, make a casting agent solution by mixing five milliliters of the diluent with four milliliters of the filtered compound. Next, add 450 microliters of the curing agent and be sure to use the casting agent solution within 20 minutes.
Mix the casting agent solution directly before use. To ensure proper viscosity, draw up the casting agent solution into a 10 milliliter syringe. Attach a blunt 20 gauge needle to the syringe and fill the needle with the solution.
Insert the needle into the left ventricle through the same hole used for flushing the blood and then secure needle into place With a hemostat clamp, slowly inject the casting agent solution manually into the left ventricle at approximately three milliliters per minute as excess pressure may lead to incorrect flow or potential vessel rupture during injection. Look for signs of successful perfusion, including visualization of the casting agent in intestine and liver vasculature, distal limb discoloration, and nose and tongue discoloration. Readjust needle if necessary to achieve proper perfusion.
Remove the brain and place in 4%paraform aldehyde overnight at four degrees Celsius. Next, dehydrate the brain over the course of seven days by placing the brain directly into increasingly concentrated ethanol at room temperature starting on the first day at 25%ethanol. Once the brain is dehydrated, clarify the brain tissue around the blood vessels by immersing the perfused brain in methyl salicylate for two days at room temperature after two days, the vasculature of the whole clarified brain can be visualized un sectioned by immersion in methyl salicylate under a bright field microscope, as well as a dissecting microscope or micro CT scan Using brightfield microscopy, the vasculature can be viewed at different focal planes without sectioning the brain, as well as individual micro vessels at higher magnification.
Shown here in the lower panel. Under a dissecting scope, one can see the entire cerebral vascular structure in one focal plane, allowing better visualization of its three dimensional structure. This image shows large vessel morphology and whole brain vasculature, but lacks the details shown in pictures taken with brightfield microscopy.
Micro CT scanning allows for 3D visualization of the vasculature and allows for construction of rotatable 3D models. To visualize the vasculature shown here is an abnormal cerebral vascular structure detected by micro CT correlating with a brightfield microscope image of the same vascular malformation. Together, these data show that the same sample can be analyzed using multiple imaging tools after vascular casting.
After watching this video, you should have a good understanding of how to cast blood vessels of the adult mouse sprain using microfill solution. This procedure includes clearing of the blood from the vasculature intraventricular injection of the microphone, solution collection and processing of the adult mouse brain and imaging of the cerebro vasculature using brightfield microscopy. The overall goal of this procedure is to view the ceal vasculature of the adult mouse brain in three dimensions.
