This study demonstrates a method for inducing hypoxia in an anesthetized mouse model to investigate glucose metabolism and structural brain changes using PET and MRI imaging techniques. The protocol outlines the steps for preparing the animal, inducing hypoxia, and acquiring imaging data to assess the effects of reduced oxygen on brain function.
Take an anesthetized mouse and provide oxygen using a nose cone.
The mouse's right common carotid artery was ligated to reduce blood flow to the right brain hemisphere, predisposing it to hypoxia-induced damage.
Insert a catheter to deliver radiolabeled glucose intravenously.
Position the mouse in a scanner to simultaneously image glucose metabolism using positron-emission tomography, or PET, and structural changes using magnetic resonance imaging, or MRI.
Induce hypoxia by reducing oxygen, thereby exacerbating cellular stress in the vulnerable hemisphere. The stress leads to intracellular water accumulation, restricting water molecule movement within swollen cells compared to the extracellular space.
In MRI images, restricted water molecule movement appears as dark areas in the affected hemisphere, indicating hypoxia-induced damage.
Simultaneously, the injected radiolabeled glucose reaches the brain and is absorbed by healthy cells.
PET imaging shows radiolabeled glucose distribution, where dark areas in the affected hemisphere indicate reduced glucose uptake due to hypoxia-induced damage.
Check the operation of the oxygen and nitrogen flow meters by first turning on the oxygen and nitrogen error sources. Then power on the flow meters. At the flow rate of 1 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 radiotracer to the animal bed within the field of view. Anesthetize the mouse with isoflurane and prewarm its tail to prepare it for catheter insertion.
Once ready, insert up to 5 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 1% to 2% isoflurane flow at between 0.5 and 1 liter per minute. Insert a rectal probe thermometer. Ensure that temperature and respiration readings are functional. Next, draw around 600 microcuries of the radiotracer dose in 200 microliters of saline into a 1 milliliter syringe and place it into a syringe pump.
Connect approximately 3 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 the rare try 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 6 millimeter by 9 millimeter.
Check the waterline width using the CalcLinewidth 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 radiotracer. 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 airflow and immediately powering on the oxygen and nitrogen flow meters to deliver 8% oxygen and 92% nitrogen.
At this point, reduce the isoflurane to 0.8%. Immediately after initiating the hypoxic challenge, begin diffusion weighted image acquisition using the previous scan setup. 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 isoflurane concentration back to 1% 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.
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