The overall goal of this procedure is to simultaneously acquire posit one emission tomography and magnetic resonance imaging data during the onset of a hypoxic ischemic injury. This is accomplished by first ligating, the common carotid artery unilaterally in a mouse. The second step is to prepare the animal for imaging and to acquire baseline PET and MRI data.
The final step is to acquire imaging data during and after a hypoxic challenge. Ultimately, simultaneous PET and MRI acquisition is used to demonstrate changes in water diffusion and in flu deoxy glucose uptake in the lesion during and after hypoxia. This method can provide insights into dynamic changes in physiology and biochemistry during stroke, but it can also be applied in other cases where the system being observed is not at steady state, but is changing.
Prepare a sterile surgical field with the tools shown here, positioned conveniently around the exterior. Turn on the heating pad and ensure it reaches 37 degrees Celsius. Before beginning the procedure.
Anesthetize the mouse using one to 3%isof fluorine, and apply ophthalmic ointment to the eyes. Place it in the supine position and confirm sedation with the toe pinch. Next, apply detory cream to the lower neck and upper chest area using one to two cotton swabs.
Wait about two minutes and then remove the hair and cream using wet gauze once the area is hair free. Sterilize the incision area with Betadine by applying it in a circular manner from inside to outside. Change into sterile surgical gloves and use surgical scissors to make a one centimeter incision along the midline of the lower neck.
Carefully separate the outer skin from the surrounding fascia with surgical scissors. Using two MCFE sin micro iris suturing forceps. Remove the connective tissue.
Go between the memory fat pads and separate the right common carotid artery from the fascia. Take care to avoid damaging veins or disturbing the vagus nerve. Next, use the forceps to exteriorize the right common carotid artery.
In a stable position, apply several drops of saline to help prevent it from drying out past two to three centimeters of six aught silk suture underneath the right common carotid artery and ligated using a double square knot. Then repeat the ligation using a second length of suture. Reposition the right common carotid artery and clean excess fluid from the opening using a sterile sponge tipped swab.
Then close the incision with six OTT silk suture and apply lidocaine topically up to seven milligrams per kilogram. Follow your institution specific guidelines for surgical pain management. Perform post-surgical monitoring during recovery until the mouse is ready for imaging.
Check the operation of the oxygen and nitrogen flow meters by first turning on the oxygen and nitrogen air sources. Then power on the flow meters at the flow rate of one liter per minute. Set the flow of oxygen to 114.3 milligrams per minute and the flow of nitrogen to 1.150 grams per minute.
Next, prepare the animal bed by ensuring that the anesthesia, respiratory pad and heater systems are positioned securely and functional. Then attach fiducial markers containing radio tracer to the animal bed within the field of view. You anesthetize the mouse with isof fluorine and prewarm its tail to prepare it for catheter insertion.
Once ready, insert up to five centimeters of a PE 10 catheter prefilled with heparinized saline. Secure the IV line at the site of insertion with a drop of cyanoacrylate adhesive. Then transfer the animal to the prepared animal bed.
Reapply ophthalmic ointment to the mouse's eyes to prevent drying and stabilize the animal's head by placing its upper incisors around the tooth bar and putting the ear bars into place. Begin one to 2%isof fluorine flow at between 0.5 and one liter per minute. Insert a rectal probe thermometer, ensure that temperature and respiration readings are functional.
Next, draw around 600 micro curies of the radiotracer dose in 200 microliters of saline into a one milliliter syringe and place it into a syringe pump. Connect approximately three meters of heparinized PE 10 tubing to the syringe and the other end to the tail vein catheter line. Check that the positioning of the MRI coil and any of the lines and cables, especially the anesthesia tubing are not entangled.
Ensure that the center of the brain is aligned with the centers of the MRI coil PET system and MRI magnet. Then carefully slide the animal bed forward into the bore of the magnet. Perform tuning and matching of the MRI coil by rotating the adjustment knobs on the coil to minimize impedance and frequency mismatches.
Next, select a rare trip pilot sequence and run the sequence from the scan control window to acquire a scout image. Check positioning of the animal and adjust its position if necessary until the brain is centered. Then reset shims to zero value.
Now run a point. Resolve spectroscopic scan sequence within the brain using a rectangular volume of 3.9 millimeters by six millimeters by nine millimeters. Check the waterline width using the calc line width macro command.
If the full width at half maximum value is acceptable, position the slice plan for the diffusion weighted imaging scan using the geometric editor when the resulting slice plan is aligned as desired. Copy this slice plan in the scan control window for all subsequent scans and begin the image acquisition. Next with the PET acquisition prepared and ready to begin, start the infusion pump After the saline from the catheter has been injected, begin the PET acquisition.
In order to capture the entry of radio tracer. Monitor the count rate and look for gradual increase in counts indicative of a successful injection. After 10 to 15 minutes, initiate the hypoxic challenge by turning off the medical air flow and immediately powering on the oxygen and nitrogen flow meters to deliver 8%oxygen and 92%nitrogen.
At this point, reduce the isof fluorine to 0.8%immediately after initiating the hypoxic challenge. Begin diffusion weighted image acquisition using the previous scan set up. Begin a second diffusion weighted image acquisition immediately after the first scan is completed.
End the hypoxic challenge by powering off the flow meters, restoring medical airflow, and returning the isof fluorine concentration back to one to 2%Acquire a post hypoxia diffusion weighted imaging scan, then turn off the infusion pump and acquire anatomical images in the axial and sagittal planes using the geometry editor. Ensure that the acquisition field of view covers the brain. Using this method, changes in diffusion are detectable rapidly after initiation of a stroke as expected.
Apparent diffusion coefficient values on the occluded side of the brain decreases as the injury progresses.Shown. Here are coronal and transverse slices of an animal showing FDG uptake. The pet image is in the foreground and is registered infused with an anatomical MRI image in the background for visualization.
Concurrent with changes in the apparent diffusion coefficient, hemispherical differences can be observed in the uptake of FDG after beginning the hypoxic challenge. In two of three cases, EPS later uptake decreased relative to contralateral uptake after hypoxia. Though this was not true, in all cases likely due to animal variability, We can use this general procedure with different kinds of MRI and pet contrast in order to observe other physiological parameters or targets in the brain.
