Name: acquisition of resting-state functional magnetic resonance imaging data in the rat
Uploaded: 2021-08-28T15:00:00.000+00:00
Duration: 12 min 41 s
Description: Watch this Scientific Journal Video about Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat at JoVE.com

This work was supported by funding from the National Institute of Health (NIH)&#39;s National Institute on Drug Abuse (NIDA) [DJW, EDKS, and EMB were supported by Grant R21DA044501 awarded to Alan I. Green and DJW was supported by Grant T32DA037202 to Alan J. Budney] and the National Institute on Alcohol Abuse and Alcoholism (NIAAA) [Grant F31AA028413 to Emily D. K. Sullivan]. Additional support was provided through Alan I. Green&#39;s endowed fund as the Raymond Sobel Professor of Psychiatry at Dartmouth.
Hanbing Lu is supported by the National Institute on Drug Abuse Intramural Research Program, NIH.
The authors wish to acknowledge and thank the late Alan I. Green. His unwavering dedication to the field of co-occurring disorders helped to establish collaboration among the authors. We thank him for his mentorship and guidance, which will be greatly missed.

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
<ol>
	<li>Subcutaneous infusion line
	<ol>
		<li>Partially remove a 23 G needle from its package so that the needle point remains sterile.</li>
		<li>Securely hold the hub of the needle and use a razor blade to score the needle shaft where it meets the hub.</li>
		<li>Clamp a needle holder around the shaft directly below the scoring and gently break the shaft from the hub.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dilution of dexmedetomidine and atipamezole
	<ol>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Scanning parameters
	<ol>
		<li>Use the parameters presented in Table 1 to prepare scanning sequences.</li>
	</ol>
	</li>
</ol>
2. Phase 1 anesthesia: Animal induction and preparation
<ol>
	<li>Setup
	<ol>
		<li>Ensure that all equipment is on and working properly including the oxygen and air mixer, heating pad, and active scavenging system (see Figure 1).</li>
		<li>Set the heating system&#39;s temperature set point to 37.5 &#176;C.</li>
	</ol>
	</li>
	<li>Animal induction
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Animal preparation
	<ol>
		<li>Apply ophthalmic lubricating ointment to each eye to prevent drying.</li>
		<li>Confirm the depth of anesthesia by a lack of toe pinch response.</li>
		<li>Use clippers to shave a 2&#34; by 2&#34; square area on the lower lumbar region of the animal&#39;s back (i.e., directly above the tail).</li>
		<li>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.</li>
		<li>Switch isoflurane flow from the preparation space to the animal cradle.</li>
		<li>Move the animal into the animal cradle. Place the rat&#39;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.</li>
		<li>Position the respiration pad under the rat&#39;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).</li>
		<li>Monitor the animal&#39;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).</li>
	</ol>
	</li>
</ol>
3. Phase 2 anesthesia: Animal setup
<ol>
	<li>Insert ear bars into the ear canal to stabilize the rat&#39;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).</li>
	<li>Insert the temperature probe into a pre-lubricated, disposable probe cover. Gently insert the temperature probe approximately &#189;&#34; into the rectum, and tape it to the base of the tail with medical tape.</li>
	<li>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 (&#60;95%).</li>
	<li>Use the rat&#39;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).</li>
	<li>Set the drug pump to eject the calculated infusion rate.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using an alcohol wipe, clean the shaved area to remove any stray hair.</li>
	<li>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.</li>
	<li>Secure the needle to the skin with a 3&#34; 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.</li>
	<li>Begin the infusion of subcutaneous dexmedetomidine.</li>
	<li>Place a piece of gauze on the bridge of the rat&#39;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&#39;s head, centering it over the brain (see Figure 3b,c).</li>
	<li>Secure all lines and cables within the animal cradle with lab tape and check whether all the physiology signals are stable (see Figure 2).</li>
	<li>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.</li>
	<li>Move the animal into the bore and tune the magnet.</li>
</ol>
4. Phase 3 anesthesia: Anatomical scan acquisition
<ol>
	<li>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.</li>
	<li>Use the FLASH localizer scan to ensure the brain is aligned with the magnet isocenter (Figure 5a). Reposition the animal and repeat if necessary.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Acquire 23 slices using both the FLASH and RARE axial protocols to aid in registration to a common space during post-scan analysis.</li>
	<li>Shim across the whole brain using the PRESS sequence.</li>
</ol>
5. Phase 4: Resting-state scan acquisition
<ol>
	<li>After completing anatomical scans, reduce isoflurane to 0.5% to 0.75%, adjusting so that the animal&#39;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.</li>
	<li>When physiology is stable (respiration range is 60-75 bpm with no gasping or irregularities, core body temperature is 37.5 &#177; 1.0 &#176;C, and oxygen saturation is 95% or greater), acquire a 15 slice EPI scan using the same slice offset as the anatomical axial series.</li>
	<li>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.</li>
	<li>Obtain at least three high-quality resting-state scans.</li>
</ol>
6. Post-scan recovery
<ol>
	<li>When scanning is complete, increase isoflurane to 2% and stop the subcutaneous dexmedetomidine infusion.</li>
	<li>Remove the animal cradle from the magnet bore, unwrap the animal, and remove ear bars, temperature probe, pulse oximeter clip, and the dexmedetomidine needle.</li>
	<li>Inject 0.015 mg/kg of the diluted atipamezole solution into the rat&#39;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).</li>
	<li>Place the rat back in the home cage on top of a heating pad and monitor until the animal is ambulatory.</li>
</ol>

<ol>
	<li>Smitha, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+state+fMRI:+A+review+on+methods+in+resting+state+connectivity+analysis+and+resting+state+networks.">Resting state fMRI: A review on methods in resting state connectivity analysis and resting state networks.</a> The Neuroradiology Journal. 30 (4), 305-317 (2017).</li><li>Gorges, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+mapping+in+the+animal+model:+Principles+and+applications+of+resting-state+fMRI.">Functional connectivity mapping in the animal model: Principles and applications of resting-state fMRI.</a> Frontiers in Neurology. 8, (2017).</li><li>Paasonen, J., Stenroos, P., Salo, R. A., Kiviniemi, V., Gr&#246;hn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+under+six+anesthesia+protocols+and+the+awake+condition+in+rat+brain.">Functional connectivity under six anesthesia protocols and the awake condition in rat brain.</a> NeuroImage. 172, 9-20 (2018).</li><li>Pawela, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+use+of+medetomidine+anesthesia+in+rats+for+extended+studies+using+task-induced+BOLD+contrast+and+resting-state+functional+connectivity.">A protocol for use of medetomidine anesthesia in rats for extended studies using task-induced BOLD contrast and resting-state functional connectivity.</a> NeuroImage. 46 (4), 1137-1147 (2009).</li><li>Jonckers, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+anesthesia+regimes+modulate+the+functional+connectivity+outcome+in+mice.">Different anesthesia regimes modulate the functional connectivity outcome in mice.</a> Magnetic Resonance in Medicine. 72 (4), 1103-1112 (2014).</li><li>Williams, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+alpha-chloralose,+medetomidine+and+isoflurane+anesthesia+for+functional+connectivity+mapping+in+the+rat.">Comparison of alpha-chloralose, medetomidine and isoflurane anesthesia for functional connectivity mapping in the rat.</a> Magnetic Resonance Imaging. 28 (7), 995-1003 (2010).</li><li>Zhurakovskaya, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+functional+connectivity+differences+between+sleep-like+states+in+urethane+anesthetized+rats+measured+by+fMRI.">Global functional connectivity differences between sleep-like states in urethane anesthetized rats measured by fMRI.</a> PloS One. 11 (5), 0155343 (2016).</li><li>Fukuda, M., Vazquez, A. L., Zong, X., Kim, S. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+&#945;2-adrenergic+receptor+agonist+dexmedetomidine+on+neural,+vascular+and+BOLD+fMRI+responses+in+the+somatosensory+cortex.">Effects of the &#945;2-adrenergic receptor agonist dexmedetomidine on neural, vascular and BOLD fMRI responses in the somatosensory cortex.</a> The European Journal of Neuroscience. 37 (1), 80-95 (2013).</li><li>Brynildsen, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+characterization+of+a+robust+survival+rodent+fMRI+method.">Physiological characterization of a robust survival rodent fMRI method.</a> Magnetic Resonance Imaging. 35, 54-60 (2017).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+brains+also+have+a+default+mode+network.">Rat brains also have a default mode network.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (10), 3979-3984 (2012).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-+but+not+high-frequency+LFP+correlates+with+spontaneous+BOLD+fluctuations+in+rat+whisker+barrel+cortex.">Low- but not high-frequency LFP correlates with spontaneous BOLD fluctuations in rat whisker barrel cortex.</a> Cerebral Cortex. 26 (2), 683-694 (2016).</li><li>Tsai, P. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Converging+structural+and+functional+evidence+for+a+rat+salience+network.">Converging structural and functional evidence for a rat salience network.</a> Biological Psychiatry. 88 (11), 867-878 (2020).</li><li>Murphy, K., Bodurka, J., Bandettini, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+long+to+scan?+The+relationship+between+fMRI+temporal+signal+to+noise+ratio+and+necessary+scan+duration.">How long to scan? The relationship between fMRI temporal signal to noise ratio and necessary scan duration.</a> NeuroImage. 34 (2), 565-574 (2007).</li><li>Birn, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+scan+length+on+the+reliability+of+resting-state+fMRI+connectivity+estimates.">The effect of scan length on the reliability of resting-state fMRI connectivity estimates.</a> NeuroImage. 83, 550-558 (2013).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronized+delta+oscillations+correlate+with+the+resting-state+functional+MRI+signal.">Synchronized delta oscillations correlate with the resting-state functional MRI signal.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (46), 18265-18269 (2007).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registering+and+analyzing+rat+fMRI+data+in+the+stereotaxic+framework+by+exploiting+intrinsic+anatomical+features.">Registering and analyzing rat fMRI data in the stereotaxic framework by exploiting intrinsic anatomical features.</a> Magnetic Resonance Imaging. 28 (1), 146-152 (2010).</li><li>Cox, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFNI:+software+for+analysis+and+visualization+of+functional+magnetic+resonance+neuroimages.">AFNI: software for analysis and visualization of functional magnetic resonance neuroimages.</a> Computers and Biomedical Research. 29 (3), 162-173 (1996).</li><li>Ash, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+with+the+retrosplenial+cortex+predicts+cognitive+aging+in+rats.">Functional connectivity with the retrosplenial cortex predicts cognitive aging in rats.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (43), 12286-12291 (2016).</li><li>Hsu, L. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+insular-frontal+networks+predict+future+nicotine+dependence+severity.">Intrinsic insular-frontal networks predict future nicotine dependence severity.</a> The Journal of Neuroscience. 39 (25), 5028-5037 (2019).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting-state+functional+MRI+reveals+altered+brain+connectivity+and+its+correlation+with+motor+dysfunction+in+a+mouse+model+of+Huntington's+disease.">Resting-state functional MRI reveals altered brain connectivity and its correlation with motor dysfunction in a mouse model of Huntington's disease.</a> Scientific Reports. 7, (2017).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abstinence+from+cocaine+and+sucrose+self-administration+reveals+altered+mesocorticolimbic+circuit+connectivity+by+resting+state+MRI.">Abstinence from cocaine and sucrose self-administration reveals altered mesocorticolimbic circuit connectivity by resting state MRI.</a> Brain Connectivity. 4 (7), 499-510 (2014).</li><li>Seewoo, B. J., Joos, A. C., Feindel, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analytical+workflow+for+seed-based+correlation+and+independent+component+analysis+in+interventional+resting-state+fMRI+studies.">An analytical workflow for seed-based correlation and independent component analysis in interventional resting-state fMRI studies.</a> Neuroscience Research. 165, 26-37 (2021).</li><li>Broadwater, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adolescent+alcohol+exposure+decreases+frontostriatal+resting-state+functional+connectivity+in+adulthood.">Adolescent alcohol exposure decreases frontostriatal resting-state functional connectivity in adulthood.</a> Addiction Biology. 23 (2), 810-823 (2018).</li><li>Jaime, S., Cavazos, J. E., Yang, Y., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observations+using+simultaneous+fMRI,+multiple+channel+electrophysiology+recording,+and+chemical+microiontophoresis+in+the+rat+brain.">Longitudinal observations using simultaneous fMRI, multiple channel electrophysiology recording, and chemical microiontophoresis in the rat brain.</a> Journal of Neuroscience Methods. 306, 68-76 (2018).</li></ol>

The authors have nothing to disclose.

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...

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 function3,4,5,6,7,8. We previously reported an anesthetic regime that applied a low dose of isoflurane along with a low dose of the &#945;2 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 network10,11 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.

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

acquisition of resting-state functional magnetic resonance imaging data in the rat

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 &#945;2 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.

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...

Watch this Scientific Journal Video about Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat at JoVE.com

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

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.

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task ...

acquisition-resting-state-functional-magnetic-resonance-imaging-data

Research

JoVE Journal

Neuroscience

4.1K Views.  Dartmouth-Hitchcock Medical Center. 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.

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Training Dogs for Awake, Unrestrained Functional Magnetic Resonance Imaging

We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for acquisition of functional and structural data. This method of training dogs for awake, unrestrained fMRI employs a generalization procedure of stationing in several dissimilar locations to facilitate transfer of the stationing behavior to the real MRI scan environment; it does so without the need for extensive training time in the MRI scan environment, which can be expensive. Further, this method splits the training of a stationing (i.e., chin rest) behavior from desensitization to the MRI environment (i.e., 100+ decibel scan audio), the latter accomplished during dedicated Auditory Exposure conditioning sessions. The complete training and testing protocol required 14 hours and resulted in immediate transfer to novel locations. We also present examples of canine fMRI data that have been acquired from visual face processing and olfactory discrimination paradigms.

Magnetic resonance imaging (MRI) on unrestrained awake dogs is a new method with several advantages over imaging with physical or chemical restraint. This protocol introduces a cost-effective training method that minimizes training in the MRI environment, which can be expensive, and maximizes the subject pool available for canine functional MRI.

We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for ...

Behavior

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Medicine

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular ...

Studying Metabolic Brain Connectivity Using 2-Deoxy-2-[18F]Fluoro-D-Glucose Dynamic Positron Emission Tomography at the Single-subject Level

To this day, metabolic brain connectivity is mostly studied on a group level through the acquisition of static positron emission tomography (PET) data of multiple subjects. Our research groups are currently studying changes in metabolic connectivity across multiple time points following an intracerebral hemorrhage on an intrasubject level in rats. To investigate intrasubject metabolic brain connectivity, temporal information of the tracer uptake in different brain regions is required, which can be achieved through dynamic PET. In this publication, we give a detailed description of our data acquisition and analysis protocol.
Dynamic PET data of the rat brain are acquired on a dedicated preclinical PET system using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) as tracer. The tracer is injected intravenously as a bolus at the start of the PET scan. During the 60 min acquisition, animals are sedated with medetomidine.
After acquisition, the PET data are reconstructed into thirty 2 min time frames using an iterative reconstruction algorithm (Maximum-Likelihood Expectation-Maximization). A parcellated atlas consisting of multiple volumes of interest (VOIs) is used to extract time-activity curves of each VOI, which are then used to calculate the Pearson correlation coefficient between each pair of VOIs.
This dynamic PET protocol enables the assessment of metabolic connectivity differences between two single scans, rather than between groups of scans. This approach allows for the study of changes in metabolic connectivity within a single subject across different time points, or for the comparison of an individual&#39;s metabolic connectivity to a normal database. Such comparisons could be useful for tracking disease progression or aiding in the diagnosis of neurological disorders characterized by disrupted communication between brain regions, such as epilepsy or dementia.

The acquisition of dynamic positron emission tomography (PET) data and reconstruction into time frames allows for metabolic brain connectivity analysis at the single-subject level. We describe a method to acquire [18F]FDG dynamic PET data of the rat brain and obtain a connectivity matrix through the extraction of time-activity curves of volumes of interest.

To this day, metabolic brain connectivity is mostly studied on a group level through the acquisition of static positron emission tomography (PET) data of ...

Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging

Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing negative findings in routine clinical imaging in the acute phase hinders early and appropriate care in these patients. It has been acknowledged that different impact parameters may affect and even accelerate the progress of subsequent neuropsychological symptoms following mTBI. However, the association of impact parameters during concussion to the outcome has not been extensively examined. In the current study, an animal model with closed-head injury (CHI) modified from the weight-drop injury paradigm was described and demonstrated in detail. Adult male Sprague-Dawley rats (n = 20) were randomly assigned to CHI groups with different impact parameters (n = 4 per group). Longitudinal MR imaging studies, including T2-weighted imaging and diffusion tensor imaging, and sequential behavioral assessments, such as modified neurological severity score (mNSS) and the beam walk test, were conducted over a 50-day study period. Immunohistochemical staining for astrogliosis was performed on day 50 post-injury. Worse behavioral performance was observed in animals following repetitive CHI compared to the single injury and sham group. By using longitudinal magnetic resonance imaging (MRI), no significant brain contusion was observed at 24 h post-injury. Nevertheless, cortical atrophy and alteration of cortical fractional anisotropy (FA) were demonstrated on day 50 post-injury, suggesting the successful replication of clinical uncomplicated mTBI. Most importantly, changes in neurobehavioral outcomes and image features observed after mTBI were dependent on impact number, inter-injury intervals, and the selected impact site in the animals. This in vivo mTBI model combined with preclinical MRI provides a means to explore brain injury on a whole-brain scale. It also allows the investigation of imaging biomarkers sensitive to mTBI across varying impact parameters and severity levels.

Here, we present a protocol to establish a closed-head injury animal model replicating the neuroimage outcome of uncomplicated mild traumatic brain injury with the preserved brain structure in the acute phase and long-term brain atrophy. Longitudinal magnetic resonance imaging is the primary method used for evidence.

Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing ...