The overall goal of this procedure is to achieve high throughput phenotyping with either live or fixed mice using multiple mouse brain MRI. This is accomplished by first anesthetizing each mouse. Next for in vivo scanning, place each mouse onto a size appropriate sled before mounting in a loading array and positioning it into the bore of the magnet.
Or for ex vivo imaging, perform trans cardiac perfusion, isolate the skull structure and place this into imaging tubes, which are then placed into a 16 channel solenoid coil array positioned in the magnet bore. Ultimately, three dimensional magnetic resonance imaging volumes can be obtained that show high quality brain images suitable for phenotyping, major and minor structures in the mouse brain. The main advantage of this technique over existing methods like histology is that it conserves the gross anatomy of the specimens still within their skulls and allows for whole brain coverage and three dimensional analysis.
This method can help answer key questions in the biomedical fields, such as the relationship between genes and anatomical distinctions that represent phenotypes in the mouse model of disease. Generally, individuals new to this technique will find the development of custom mouse loading and radio frequency coiler arrays required for high throughput brain imaging. The most challenging Before beginning this protocol, obtain approval for all mass handling procedures from your institution's, local animal care committee, institutional animal care and use committee, or equivalent first procedures such as identification and weight.
Recordings of the animals should be performed under a biological safety cabinet. Next, transfer the animals to an autoclavable plastic container for transport to the MRI induction chamber. Anesthetize the animals to be scanned in the prewarm induction chamber using 4%ISO fluorine and four liters per minute of oxygen.
Animals are fully anesthetized once they fail to respond to paw pinch. Fur from the chest can be removed using a hair remover such as nare if necessary, to provide better contact with the ECG and temperature monitoring devices that have been built into the custom sled. Also apply ISV such as tears natural PMM to the eyes to prevent drying and administer approximately 0.3 milliliters of saline via subcutaneous injection.
To hydrate the animals. Now load the mice onto individual sleds with embedded ECG respiration and temperature probes. Be careful to position the front and back appendages into the depressions found on the sleds.
In instances when ECG monitoring is required, make sure that the hair free regions on the underside of the mouse line up with the ECG probes, immobilize each animal with a head strap and nose strap, and then slide each sled into an open-ended 50 milliliter conical tube. Up to seven live mice can be prepared for scanning in one imaging session. Next, mount the conical tubes onto the loading array.
Make sure to connect all of the physiological monitoring lines once all animals are loaded. Set the isof fluorine level in the magnet bore to 2%and the oxygen level to eight liters per minute. Now transport the loading array to the magnet and position it on the rail system.
Use the rail system to couple the loading array with the mouse hive, which is designed to position mice uniformly into each radio frequency coil positioned in the center of the magnet bore. When fully inserted into the magnet, the centrifuge tubes dock onto the anesthetic delivery system within the RF coils as seen here, ISOF fluorine mixed with oxygen is supplied from the mouse hive and to the specimen through a tube along the axis of each individual coil. This anesthetic gas mixture flows into the tubes past the mice and is collected by an active scavenging unit attached to the back of the loading array.
Depending on the physiological readings of the animals, such as respiration and temperature, isof, fluorine levels should be set between 0.9 and 2%Now, scan the animals using a fast bin echo sequence. The duration of each three dimensional scan is approximately three hours with a resulting image resolution of 125 micrometers. Be sure to monitor ECG readings, respiration, and temperature on each animal throughout the course of the scan.
Animals should also be kept warm during the scanning session with a flow of warmed air. When the scan is complete, remove the loading array from the magnet and place it in a warm induction chamber filled with 100%oxygen. Unload the animals from the tubes and transfer them into plastic sealed containers for transport to a biological safety cabinet.
Finally, place the animals in a warm draft free cage and allow them to recover from anesthesia for exvivo animal imaging. First, fully anesthetize the animals using an intraperitoneal injection of a combination of ketamine and xylazine. Before beginning perfusion, assess the surgical plane of anesthesia by toe or paw pinch.
Open the chest cavity and insert a needle or safety winged infusion set into the left ventricle of the heart. Then cut the right oracle to begin trans cardiac perfusion. First flush with 30 milliliters of room temperature, one x phosphate buffered saline mixed with one microliter per milliliter heparin and two millimolar pro hands using a flow rate of approximately 100 milliliters per hour.
This should take about 20 minutes. Next fixation pass with 30 milliliters of room temperature, 4%paraform aldehyde mixed with two millimolar pro hands at 100 milliliters per hour. Now decapitate the animal and remove the skin, lower jaw ears, and the cartilaginous nose tip.
Then place the remaining skull structure in 4%para formaldehyde mixed with two millimolar pro hands and leave overnight at four degrees Celsius. The next day, transfer the specimen to a solution of one x phosphate buffered saline, mixed with 0.02%sodium azide with two millimolar pro hands for preservation. High resolution three three-dimensional MRI scanning at seven Tesla should occur at least four days, but not longer than two and a half months.
After perfusion. Place the brains to be imaged in a 16 channel solenoid coil array and place this in the scanner. Now scan the specimens using a fast spin echo sequence.
This scan duration is approximately 12 hours, and the final images will have an isotropic resolution of 32 micrometers. After the scan is completed, remove the specimens from the imaging coil and place each skull in a 10%formalin solution with two millimolar pro hands for preservation representative in vivo. Multiple mouse brainin images from one three hour scan are shown here.
This figure shows three viewing axes for each of the seven mice scanned.Displayed. Here is a representative ex vivo brain image at 32 micron isotropic resolution. And here we can see a 3D rendering of an ex vivo image volume Once mastered.
This in vivo technique can be done in three to four hours if performed properly. This Technique can be used by researchers in many biomedical fields to explore diseases such as lupus, Huntington's, and Alzheimer's disease in the mouse model. After watching this video, you should have a good understanding of how to image both live and fix most subjects to obtain high quality brain images using MRI.
Thanks for watching.
