All protocols and procedures were carried out in compliance with federal regulations and guidelines of the Medical College of Wisconsin’s Institutional Animal Care and Use Committee (IACUC). Six Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) (300–400 g) underwent a procedure that denervated the right forepaw followed by fMRI and fcMRI studies of the visual and sensorimotor systems. Six Sprague-Dawley rats were used as a control in the study of BOLD fMRI activation in response to forepaw and radial nerve stimulation. Twenty healthy Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) (300–400 g) were used as a control in the fcMRI sensorimotor system experiments, and 15 healthy Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) (300–400 g) were used as a control in the study of the visual system.
Denervation procedure and recovery
Rats were initially anesthetized with 2.5% isoflurane (Halocarbon Laboratories, River Edge, New Jersey, USA) and placed supine on a heated surgical table. Isoflurane anesthesia was reduced to 1.5% for maintenance. An incision was made along the medial aspect of the right upper extremity. The subcutaneous tissues were retracted, and the pectoralis major muscle identified and extended into the axilla and a portion of the flank. The pectoralis was retracted medially to expose the brachial plexus and artery. There are four major nerves of the brachial plexus: median, ulnar, radial, and musculocutaneous. The largest and most visible nerve is the median. The ulnar nerve resides parallel and medial to the median nerve. The radial nerve lies deep to the brachial artery. The long and short heads of the biceps brachii are lateral to the major neurovascular bundle. The long head of the biceps brachii was reflected laterally, exposing the musculocutaneous nerve between the retracted biceps muscles. All four major nerves in the right forelimb (median, ulnar, radial, and musculocutaneous) were surgically transected. A section of each nerve was completely removed to prevent reattachment. The initial incision was then sutured and the animal was allowed to recover. Buprenorphine (0.05 mg/kg) was injected subcutaneously every 12 hours for two days for pain management. A quarter section of a 100 mg tramadol pill (ULTRAM, ORTHO-McNEIL, Titusville, NJ, USA) was crushed and added to a small amount of fruit-flavored Jell-O for treatment of phantom limb pain. The Jell-O mixture was fed to the rat each day during the recovery period. A two-week period was allowed between the denervation procedure and the scanning session. No signs of self-mutilation were found during the recuperation phase.
Surgical protocol prior to fMRI/fcMRI experiments
All rats in this study underwent a pre-imaging surgical protocol. Subject rats were removed from caging and placed in a gas anesthesia box. Isoflurane was started at 2.5% and the rat anesthetized. The rat was then placed on a heated surgical table and isoflurane continued at 1.5%. The right femoral vein was cannulated for experimental access with PE-50 tubing (Stoelting, Wood Dale, IL, USA). A tracheostomy was performed for mechanical ventilation during the fMRI protocol. Both incisions were carefully sutured, and the subject rat was placed on a custom-built, heated G-10 fiberglass rodent cradle for imaging. G-10 was used because it has a magnetic susceptibility similar to air. Animal temperature was maintained at 37°C by a recirculating water pump (Meditherm-III, Gaymar Industries, Orchard Park, NY, USA). An intravenous infusion mixture of 100 μg/kg/hr medetomidine hydrochloride (Domitor, Pfizer Animal Health, New York, NY, USA) and 2 mg/kg/hr pancuronium bromide (Hospira, Lake Forest, IL, USA) was started and isoflurane anesthesia ended. Infusion was possible in the MRI environment with the use of an MRI-compatible infusion pump (PHD 2200, Harvard Apparatus, Boston, MA, USA). Bipolar copper-beryllium electrodes were placed between the webspaces of the second and fifth digits of each forepaw.
During the pre-imaging surgery, the six denervated and six control rats underwent an additional protocol that implanted an electrode on the left radial nerve. An incision was made along the median aspect of the left upper forelimb and the brachial plexus nerves exposed. A 150 μm electrode (Plastics One, Roanoke, VA, USA) was attached to the radial nerve and the incision closed with nylon suture. A shielded MRI-compatible cable was attached from the electrode to the electrical stimulator outside the scanner.
A 9.4 T small-animal scanner (AVANCE, Bruker, Billerica, MA, USA) was used for all imaging procedures. A surface receive coil (T9208, Bruker, Billerica, MA, USA) and a linear transmit coil (T10325, Bruker, Billerica, MA, USA) were employed during the fcMRI/fMRI protocol. A rapid acquisition with relaxation enhancement (RARE) anatomic image was obtained prior to each fcMRI or fMRI experiment. For the sensorimotor system, RARE parameters were TR = 2.5 sec, TE = 50.8 ms, FOV = 35 mm, and 256 × 256 matrix. Ten 1-mm-thick contiguous slices were acquired, with the third slice located directly over the anterior commissure (−0.36 mm from bregma). For the visual system, RARE parameters were TR = 2.5 sec, TE = 50.8 ms, FOV = 35 mm, and 256 × 256 matrix. Fifteen 1-mm-thick contiguous slices were acquired, with the third slice located directly over the anterior commissure (−0.36 mm from bregma). Two resting-state fcMRI acquisitions were performed prior to each stimulation fMRI experiment. T2*-weighted echo-planar imaging (EPI) sequences were used for all fMRI/fcMRI experiments. EPI parameters for both task-activation and resting-state scans were TR = 2 sec, TE = 18.76 ms, FOV = 35 mm, 96 × 96 matrix (zero filled to 128 × 128 matrix), with the same slice geometry as the RARE images. 110 images were obtained during each fcMRI/fMRI experiment for a total acquisition time of 3 min 40 sec.
fMRI electrical stimulation method
A square-wave electrical generator (S88, Grass Telefactor, Warrick, RI, USA) equipped with a constant-current power supply (CCU, Grass Telefactor, Warrick, RI, USA) was used for stimulation. A standard block design stimulation protocol of 40 sec rest followed by three periods of 20 sec ON and 40 sec OFF was used (total of 3 min 40 sec). Electrical stimulation was computer-controlled and triggered by a transistor-transistor logic (TTL) pulse from the scanner. The forepaw was stimulated with square-wave pulses at 10 Hz frequency, 3 ms pulse width, and 2 mA amplitude, and the radial nerve stimulated at 5 Hz frequency, 1 ms pulse width, and 1 mA amplitude. Two stimulation fMRI experiments were performed for each condition for a total of four fMRI acquisitions.
fMRI visual stimulation method
Blue, MRI-compatible light-emitting diodes (LEDs) with a wavelength of 465 nm were placed bilaterally 2 cm from the eyes of the rat. The LEDs were computer-controlled (Labview Software, National Instruments, Austin, Texas, USA) and triggered by a TTL pulse from the scanner. The lights in the scanner suite were turned off. The same boxcar stimulation sequence used for the forepaw was used for visual stimulation. Light activation was used as an internal control. The BOLD activation patterns in response to visual stimulation were in good agreement with our earlier work (Pawela et al. 2008
Physiological monitoring during MRI acquisition
Several physiological parameters were continuously monitored during all imaging experiments. These included pulse oximetry (Model 8600 V, Nonin Medical, Plymouth, MN, USA), chest respiration (Model 1025, SA Instruments, Stony Brook, NY, USA), and end tidal gases (POET IQ2, Criticare Systems, Waukesha, WI, USA). The subject rat was mechanically ventilated using an MRI-compatible ventilator (MRI-1, CWE, Ardmore, PA, USA) with a 30/70 O2/N2 inhalation gas mixture. The respiratory rate was maintained between 55 and 65 breaths per minute. Respiratory parameters were adjusted to maintain physiologic parameters in normal ranges.
fMRI data analysis
All EPI acquisitions were registered to an “ideal” RARE image using the Oxford Center for Functional Magnetic Resonance of the Brain’s (FMRIB) Linear Image Registration Tool (FLIRT) program (Jenkinson and Smith 2001
). The “ideal” RARE image was chosen to have the best gray-white contrast from the entire experimental subset. Further analysis was done using the program Analysis of Functional Neuroimages (AFNI) (Cox and Hyde 1997
). The registered EPI acquisitions were averaged using the AFNI 3dcalc program. These averaged EPI datasets were used to create activation maps by performing an F-test on the time series with the block design as the only regressor (AFNI, 3dDeconvolve). A p-value of 0.005 was used for plotting and was the threshold for activation used in the time-course analysis. All activation maps were overlaid on the “ideal” RARE image for display. Time-course analysis was performed by averaging activated voxel time-courses from each specific ROI across every animal.
Regions of interest
Brain regions were drawn on the “ideal” RARE image freehand using AFNI. The Paxinos’ stereotactic rat brain atlas (Paxinos 2005
) was consulted for accuracy. Regions of interest (ROI) drawn from the sensorimotor region include the primary/secondary motor cortex (M1/M2), the primary sensory forelimb region (S1FL), the primary sensory trunk region (S1Tr), the secondary sensory region (S2), the corpus callosum (CC), the sensorimotor thalamus (SMT), the ventral posterior thalamic nucleus (VP), the posterior thalamic nucleus (VP), the reticular thalamic nucleus (Rt), the caudate putamen (CP), and the globus pallidus (GP). ROIs drawn from the visual system include the primary visual cortex (V1), the secondary visual cortex (V2), the temporal association cortex (TeA), the dorsal lateral geniculate nucleus (DLG), the lateral posterior nucleus (LP), and the superior colliculus. An ROI was also drawn for the rat hippocampus (HIP).
fcMRI seed voxel analysis
Both resting-state fcMRI experiments for each animal were combined to build a new dataset. We used cross-correlation analysis between the average time-courses of six reference voxels at the center of the region chosen from the functional anatomy. Those time-courses were correlated with every other voxel time-course in that slice. Since we are only interested in the temporal correlation due to slow periodic spontaneous oscillations, a finite impulse response filter was used to filter the high-frequency components from each of the datasets. Because of the short data size, filter parameters were adjusted to minimize the generation of artifactual frequencies (sidelobes). All voxels that passed a correlation coefficient threshold of 0.35 were considered significant, and those locations were noted.
fcMRI regional analysis
A regional pairwise correlation coefficient (RPCC) matrix was tabulated using the averaged filtered time-course from each region. The two resting-state fcMRI datasets for each individual animal were combined to create a new, 220-time-point dataset. The new dataset was used in the RPCC analysis. Datasets were first detrended to eliminate linear drifts. A low-pass filter with a cutoff at 0.1 Hz was applied to all regional time-courses. Principal component analysis (PCA) was carried out for each region within the sensorimotor system. Using PCA, a dataset of orthogonal waveforms was determined for each region. Briefly, PCA is a multivariate technique that replaces the measured variables by a set of principal components arranged in the order of decreasing standard deviation (SD) or energy distribution. For this study, the first two components accounted for more than 75% of the SD for each ROI, and a mixture of the two was therefore used in the RPCC analysis. The resulting time series for each region was correlated with every other regional time component to obtain a pair-wise correlation matrix. The PCA was run independently for each region.