Resting-state functional magnetic resonance imaging, or resting-state fMRI, has become an increasingly popular method to study brain function in a resting non-task state. Technological advances have allowed resting-state fMRI to be adapted for use in rodent models, and can be used to uncover mechanisms underlying disease states. The protocol described here combines low-dose isoflurane with low dose dexmedetomidine, allowing for high quality data acquisition and preservation of brain network function.
This method also allows for spontaneous breathing and near-normal physiology for up to five hours. In this video, we cover monitoring of the rat's physiology during four distinct phases of anesthesia, animal induction and preparation, animal setup, anatomical scan acquisition, and resting-state scan acquisition followed by quality assessment of the data. Using a system to deliver inhaled anesthetic as well as scavenge waste gases from all needed areas, induce anesthesia with 2.5%isoflurane in 30%oxygen-enriched air.
Once the animal is anesthetized, remove it from the chamber, weigh the animal, and place it in the nose cone on the heating pad in the preparation space, remaining at 2.5%isoflurane. Apply ophthalmic lubricating ointment to each eye to prevent drying. Confirm the depth of anesthesia by a lack of toe pinch response, Use clippers to shave a two-inch by two-inch square area on the lower lumbar region of the animal's back directly above the tail.
Administer 0.015 milligrams per kilogram of the dexmedetomidine solution with an intraperitoneal injection into the lower-right quadrant of the abdomen using a 25-gauge needle. Switch isoflurane flow from the preparation space to the animal cradle. Move the animal into the animal cradle.
Place the rat's front teeth securely over and into the bite bar. Push the nose cone over the nose to ensure a tight fit. If the nose cone does not cover the lower jaw, Parafilm can be used to gently hold the jaw closed while also sealing around the nose cone.
Position the respiration pad under the animal's abdomen below the rib cage and reposition it until the respiration waveform shows a deep trough centered on each breath. Move to the next phase of anesthesia when respiration is less than 40 breaths-per-minute. Insert that ear bars into the ear canal to stabilize the rat's head in the animal cradle.
Once positioned, pull forward on the bite bar and confirm the head does not move. Readjust the nose cone and Parafilm as needed. Insert the temperature probe into a pre-lubricated disposable probe cover.
Gently insert the temperature probe approximately 1/2 inch into the rectum and tape it to the base of the tail with medical tape. Place the pulse oximeter clip onto the metatarsal area of the hind foot, ensuring the light source is on the bottom of the foot. Rotation of the clip can affect the signal, thus, creating a holder to keep the paw and clip upright will lead to greater stability.
Calculate the infusion rate for 0.015 milligrams per kilogram per hour of dexmedetomidine. Set the drug pump to the calculated infusion rate and fill the infusion line. Using an alcohol wipe, clean the shaved area to remove any stray hair.
Pinch the skin approximately two finger widths above the base of the tail. Insert 1/3 of the infusion line needle into the tented skin. Secure the needle to the skin with a three-inch piece of wide medical tape.
Place a second piece of wide medical tape over the first across the rat and attached to both sides of the animal cradle. It is critically important that the ferromagnetic needle is well-secured to prevent movement during the scan. Begin the infusion of subcutaneous dexmedetomidine.
Place a piece of gauze on the bridge of the rat's nose to create a level surface for the coil. Use paper tape, which does not interfere with the MRI signal, to secure the coil to the rat's head, centering it over the brain. Place paper towels over the animal, securing them to the animal cradle with laboratory tape.
If using an air heating system, wrap a plastic sheet around the entire cradle to contain the warm air. Move the animal into the bore and tune the magnet. Reduce isoflurane to 1.5%resulting in a steady increase in respiration to approximately 45 to 50 breaths-per-minute.
Remain at this level for the duration of the anatomical scanning. Use the FLASH localizer scan to ensure the brain is aligned with the magnet isocenter. Reposition the animal and repeat if necessary.
Run the higher resolution RARE localizer scan, and use this scan output to align 15 sagittal slices centered across the brain. Using the middle sagittal slice, align the center axial slice to the decussation of the anterior commissure, which appears as a dark spot. Note the slice offset to use later in the resting-state scans.
Acquire 23 slices using both the FLASH and RARE axial protocols to aid in registration to a common space during post-scan analysis. Shim across the whole brain using the PRESS sequence. After completing anatomical scans, reduce isoflurane to 0.5 to 0.75%adjusting so that the animal's respiration is 60 to 65 breaths-per-minute.
Remain at this level for at least 10 minutes before beginning resting-state scanning to ensure stability. When physiology is stable, acquire a 15 slice EPI scan using the same slice offset as the anatomical axial series. After each resting-state scan is complete, check the quality using an independent component analysis to decompose the data into spatial and temporal components.
Obtain at least three high-quality resting-state scans. When scanning is complete, increase isoflurane to 2%and stop the subcutaneous dexmedetomidine infusion. Remove the animal cradle from the magnet bore and return the animal to the preparation space.
Inject 0.015 milligrams per kilogram of the diluted atipamezole solution into the rat's hind leg muscle using a 25-gauge needle. Place the rat back in the home cage on top of a heating pad, and monitor until the animal is ambulatory. Using an independent component analysis, assess stability following each resting-state scan.
Shown here is an example of very high stability. Note that, spatially, the components have high regionality. Within the time course shown in the middle panel, the signal is stable and not predictable, which is indicative of true brain activity.
The power spectrum at the bottom shows predominantly low frequencies. Shown here is a noise component from the same scan. Note the non-regionality, high-frequency time course, and single high-frequency peak in the power spectrum.
Noise components should be de-noised during post-scan analysis. Finally, this component is from a scan where anesthesia was not stable. The time course displayed is variable and irregular.
When this happens, improvements are needed to the anesthetic protocol, commonly to the ceiling of the nose cone and the scavenging of waste gases. Stability of the animal, both physically and physiologically, is key to obtaining high-quality resting-state data. It is imperative that the animal meets the physiological thresholds established before moving to the next phase of anesthesia.
Following this survival technique, resting state fMRI can be used in longitudinal studies, in conjunction with experimental manipulations, and to explore disease models. The combination of low dose isoflurane with dexmedetomidine utilized in this protocol allows for a wide variety of preclinical studies for investigators interested in imaging the rodent brain in its resting state.
