Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat

Summary

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

Abstract

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task state. This protocol describes a preclinical survival method for obtaining rs-fMRI data. Combining low dose isoflurane with continuous infusion of the α₂ adrenergic receptor agonist dexmedetomidine provides a robust option for stable, high-quality data acquisition while preserving brain network function. Furthermore, this procedure allows for spontaneous breathing and near-normal physiology in the rat. Additional imaging sequences can be combined with resting-state acquisition creating experimental protocols with anesthetic stability of up to 5 h using this method. This protocol describes the setup of equipment, monitoring of rat physiology during four distinct phases of anesthesia, acquisition of resting-state scans, quality assessment of data, recovery of the animal, and a brief discussion of post-processing data analysis. This protocol can be used across a wide variety of preclinical rodent models to help reveal the resulting brain network changes that occur at rest.

Introduction

Resting-state functional magnetic resonance imaging (rs-fMRI) is a measure of the blood-oxygen-level-dependent (BOLD) signal when the brain is at rest and not engaged in any particular task. These signals can be used to measure correlations between brain regions to determine the functional connectivity within neural networks. rs-fMRI is widely used in clinical studies due to its non-invasiveness and the low amount of effort required of patients (as compared to task-based fMRI) making it optimal for diverse patient populations¹.

Technological advances have allowed rs-fMRI to be adapted for use in rodent models to uncover mechanisms underlying disease states (see reference² for review). Preclinical animal models, including disease or knockout models, allow a wide range of experimental manipulations not applicable in humans, and studies can also make use of post-mortem samples to further enhance experiments². Nevertheless, due to the difficulty in both limiting motion and mitigating stress, MRI acquisition in rodents is traditionally performed under anesthesia. Anesthetic agents, depending on their pharmacokinetics, pharmacodynamics, and molecular targets, influence brain blood flow, brain metabolism, and potentially affect neurovascular coupling pathways.

There have been numerous efforts to develop anesthetic protocols that preserve neurovascular coupling and brain network function^3,4,5,6,7,8. We previously reported an anesthetic regime that applied a low dose of isoflurane along with a low dose of the α₂ adrenergic receptor agonist dexmedetomidine⁹. Rats under this method of anesthesia exhibited robust BOLD responses to whisker stimulation in regions consistent with established projection pathways (ventrolateral and ventromedial thalamic nuclei, primary and secondary somatosensory cortex); large-scale resting-state brain networks, including the default mode network^10,11 and salience network¹² have also been consistently detected. Furthermore, this anesthetic protocol allows for repeated imaging on the same animal, which is important for monitoring the disease progression and the effect of experimental manipulations longitudinally.

In the present study, we detail the experimental setup, animal preparation, and physiological monitoring procedures involved. In particular, we describe the specific anesthetic phases and acquisition of scans during each phase. Data quality is assessed following each resting-state scan. A brief summary of post-scan analysis is also included in the discussion. Laboratories interested in uncovering the potential of using rs-fMRI in rats will find this protocol useful.

Protocol

All experiments were performed on a 9.4 T MRI scanner, and were approved by the Institutional Animal Care and Use Committee at Dartmouth College. Additional approval was obtained to record and show the animals used in the video and figures below.

1. Preparations before scanning

1. Subcutaneous infusion line
   1. Partially remove a 23 G needle from its package so that the needle point remains sterile.
   2. Securely hold the hub of the needle and use a razor blade to score the needle shaft where it meets the hub.
   3. Clamp a needle holder around the shaft directly below the scoring and gently break the shaft from the hub.
   4. Insert 1/3 of the needle shaft (blunt end) into previously sterilized PE50 line with enough line length to extend from the drug pump to the animal inside the magnet bore.
2. Dilution of dexmedetomidine and atipamezole
   1. Prepare a solution of diluted dexmedetomidine hydrochloride using 0.5 mL of 0.5 mg/mL stock mixed with 9.5 mL of sterile saline in a clear, sterile glass bottle (diluted concentration = 0.025 mg/mL).
   2. Prepare a solution of diluted atipamezole using 0.1 mL of 5 mg/mL stock mixed with 9.9 mL of sterile saline in a clear, sterile glass bottle (diluted concentration = 0.05 mg/mL).
3. Scanning parameters
   1. Use the parameters presented in Table 1 to prepare scanning sequences.

2. Phase 1 anesthesia: Animal induction and preparation

1. Setup
   1. Ensure that all equipment is on and working properly including the oxygen and air mixer, heating pad, and active scavenging system (see Figure 1).
   2. Set the heating system's temperature set point to 37.5 °C.
2. Animal induction
   1. Place the animal (90-day old, male Sprague Dawley rat) in the induction chamber and induce anesthesia with 2.5% isoflurane in 30% oxygen-enriched air.
      NOTE: A wide range of animal ages and both sexes can be used.
   2. Once the animal is anesthetized, remove it from the chamber, weigh the animal, and place it in the nose cone (at 2.5% isoflurane) on the heating pad in the preparation space.
3. Animal preparation
   1. Apply ophthalmic lubricating ointment to each eye to prevent drying.
   2. Confirm the depth of anesthesia by a lack of toe pinch response.
   3. Use clippers to shave a 2" by 2" square area on the lower lumbar region of the animal's back (i.e., directly above the tail).
   4. Administer 0.015 mg/kg of the dexmedetomidine solution with an intraperitoneal (i.p.) injection (e.g., a 300 g rat would receive 0.18 mL) into the lower right quadrant of the abdomen using a 25 G needle.
   5. Switch isoflurane flow from the preparation space to the animal cradle.
   6. 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.
      NOTE: If the nose cone does not cover the lower jaw, use a paraffin film to gently hold the jaw closed while also sealing around the nose cone.
   7. Position the respiration pad under the rat's abdomen below the rib cage and re-position it until the respiration waveform shows a deep trough centered on each breath (see respiration waveform in Figure 2).
   8. Monitor the animal's breathing using the physiology monitoring software. Move to the next phase of anesthesia when respiration is less than 40 breaths/min (bpm; approximately 5 min after dexmedetomidine injection).

3. Phase 2 anesthesia: Animal setup

1. Insert 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. Re-adjust the nose cone and paraffin film as needed (see Figure 3a).
2. Insert the temperature probe into a pre-lubricated, disposable probe cover. Gently insert the temperature probe approximately ½" into the rectum, and tape it to the base of the tail with medical tape.
3. Place the pulse oximeter clip onto the metatarsal area of the hind foot, ensuring the light source is on the bottom of the foot (palm).
   NOTE: Rotation of the clip can affect the signal; thus, creating a holder to keep the paw and clip upright will lead to greater stability. Also note that until the rat is at normal body temperature, the oxygen saturation may be low (<95%).
4. Use the rat's weight to calculate the infusion rate to eject 0.015 mg/kg/h of dexmedetomidine (a 300 g rat receives 0.18 mL/h).
5. Set the drug pump to eject the calculated infusion rate.
6. Fill a 3 mL syringe with the sterile, diluted dexmedetomidine solution and insert the tip of the needle into the open end of the sterilized infusion line (extending from the drug pump to the animal cradle with the subcutaneous needle previously attached). Fill the line and secure the syringe in the syringe holder of the drug pump.
7. Move the pusher block forward until it touches the plunger, and the drug is expelled at the needle, ensuring the infusion line is completely filled.
8. Using an alcohol wipe, clean the shaved area to remove any stray hair.
9. 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.
10. Secure the needle to the skin with a 3" 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 (see Figure 4).
    NOTE: It is critically important that the ferromagnetic needle is well secured to prevent movement during the scan.
11. Begin the infusion of subcutaneous dexmedetomidine.
12. 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 (see Figure 3b,c).
13. Secure all lines and cables within the animal cradle with lab tape and check whether all the physiology signals are stable (see Figure 2).
14. 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.
15. Move the animal into the bore and tune the magnet.

4. Phase 3 anesthesia: Anatomical scan acquisition

1. Reduce isoflurane to 1.5%, resulting in a steady increase in respiration to approximately 45-50 bpm. Remain at this level for the duration of the anatomical scanning.
2. Use the FLASH localizer scan to ensure the brain is aligned with the magnet isocenter (Figure 5a). Reposition the animal and repeat if necessary.
3. Run the higher resolution RARE localizer scan and use this scan output to align 15 sagittal slices centered across the brain (left to right, Figure 5b).
4. Using the middle sagittal slice, align the center axial slice to the decussation of the anterior commissure, which appears as a dark spot (Figure 5c). Note the slice offset to use later in the resting-state scans.
5. Acquire 23 slices using both the FLASH and RARE axial protocols to aid in registration to a common space during post-scan analysis.
6. Shim across the whole brain using the PRESS sequence.

5. Phase 4: Resting-state scan acquisition

1. After completing anatomical scans, reduce isoflurane to 0.5% to 0.75%, adjusting so that the animal's respiration is 60-65 breaths per minute. Remain at this level for at least 10 min before beginning resting-state scanning to ensure stability.
2. When physiology is stable (respiration range is 60-75 bpm with no gasping or irregularities, core body temperature is 37.5 ± 1.0 °C, and oxygen saturation is 95% or greater), acquire a 15 slice EPI scan using the same slice offset as the anatomical axial series.
3. After each resting-state scan is complete, check the quality using an independent component analysis (ICA) to decompose the data into spatial and temporal components.
4. Obtain at least three high-quality resting-state scans.

6. Post-scan recovery

1. When scanning is complete, increase isoflurane to 2% and stop the subcutaneous dexmedetomidine infusion.
2. Remove the animal cradle from the magnet bore, unwrap the animal, and remove ear bars, temperature probe, pulse oximeter clip, and the dexmedetomidine needle.
3. Inject 0.015 mg/kg of the diluted atipamezole solution into the rat's hind leg muscle using a 1 mL syringe with a 25 G needle (i.e., a 300 g rat would receive 0.09 mL).
4. Place the rat back in the home cage on top of a heating pad and monitor until the animal is ambulatory.

Results

Following each resting-state scan, stability is assessed using an independent component analysis (ICA; example script included in Supplementary Files). Figure 6 shows examples of component outputs from resting-state scans. Figure 6a shows a signal component from a scan with high stability. Note that spatially, the component has high regionality. Within the time course below the spatial component, the signal is stable and not predictable, indicat...

Discussion

Stability of the animal, both physically and physiologically, is key to obtaining high-quality resting-state data. This protocol achieves stability by moving through four distinct phases of anesthesia. It is imperative that the animal has met the set physiological thresholds before moving to the next phase of anesthesia; since this method relies on physiological autoregulatory mechanisms, individual animals may require slightly different amounts of time at each anesthesia phase. It is our experience that taking more time...

Materials

Name	Company	Catalog Number	Comments
9.4T MRI	Varian/Bruker		Varian upgraded with Bruker console running Paravision 6.0.1 software
Air-Oxygen Mixer	Sechrist	Model 3500CP-G
Analysis of Functional NeuroImages (AFNI)	NIMH/NIH	Version AFNI_18.3.03	Freely available at: https://afni.nimh.nih.gov/
Animal Cradle	RAPID Biomedical	LHRXGS-00563	rat holder with bite bar, nose cone and ear bars
Animal Physiology Monitoring & Gating System	SAII	Model 1025	MR-compatible system including oxygen saturation, temperature, respiration and fiber optic pulse oximetry add-on
Antisedan (atipamezole hydrochloride)	Patterson Veterinary	07-867-7097	Zoetis, Manufacturer Item #10000449
Ceramic MRI-Safe Scissors	MRIequip.com	MT-6003
Clippers	Patterson Veterinary	07-882-1032	Wahl touch-up trimmer combo kit, Manufacturer Item #09990-1201
Dexmedesed (dexmedetomidine hydrochloride)	Patterson Veterinary	07-893-1801	Dechra Veterinary Products, Manufacturer Item#17033-005-10
Digital Rectal Thermometer Covers	Medline	MDS9608
FMRIB Software Library	FMRIB	MELODIC Version 3.15	Freely available at: https://fsl.fmrib.ox.ac.uk/fsl/fslwiki
Heating Pad	Cara Inc.	Model 50
Hemostat forceps, straight	Kent Scientific	INS750451-2
Isoflurane	Patterson Veterinary	07-893-1389	Patterson Private Label, Manufacturer Item #14043-0704-06
Isoflurane Vaporizer	VetEquip Inc.	911103
Lab Tape, 3/4"	VWR International	89097-990
Needles, 23 gauge	BD	305145	plastic hub removed
Parafilm Laboratory Film	Patterson Veterinary	07-893-0260	Medline Industries Inc., Manufacturer Item #HSFHS234526A
Planar Surface Coil	Bruker	T12609	2cm
Polyethylene Tubing	Braintree Scientific	PE50 50FT	0.023" (inner diameter), 0.038" (outer diameter)
Puralube Ophthalmic Ointment	Patterson Veterinary	07-888-2572	Dechra Veterinary Products, Manufacturer Item #211-38
Sprague Dawley Rats	Charles River	400 SAS SD
Sterile 0.9% Saline Solution	Patterson Veterinary	07-892-4348	Aspen Vet, Manufacturer Item #14208186
Sterile Alcohol Prep Pads	Medline	MDS090735
Surgical Tape, 1" (3M Durapore)	Medline	MMM15381Z	3M Healthcare, "wide medical tape"
Surgical White Paper Tape, 1/2" (3M Micropore)	Medline	MMM15300	3M Healthcare
Syringes, 1 mL w/ 25 gauge needle	BD	309626
Syringes, 3 mL	BD	309657
Vented induction and scavenging system	VetEquip Inc.	942102	2 liter induction chamber with active scavenging
		411724	omega flowmeter
		931600	scavenging cube, "vacuum"
		921616	nose cone, non-rebreathing