Description of the developed system architecture
The patented system (EPS 17195244.3–1666, October 2017) consists of two modules (Fig. 1), the Main unit and the MRI unit (Fig. 1), connected by optical fiber via wave guides into the MRI scanner room. The Lead Unit is placed outside the scanner room and coupled to a computer, which enables the programming of stimulus settings and fMRI design by use of E-prime® (Psychology Software Tools, Sharpsburg, Pennsylvania, USA). Also, it is connected to the MRI scanners trigger unit which serves to timely initiate the stimulation sequence at the start of the scan. The Lead unit consists of the following: Signal generator to produce the voltage-driven electric stimulation signal; oscilloscope monitors to record the electric stimulation signal transmitted back from MRI unit for quality assurance (QA) and; and two optical modules used for transmitting the electric stimulation signal and QA signals to and from the MRI unit. The MRI unit has two corresponding optical modules and optical signal converters. The electronics in the MRI scanner room are shielded by use of caging and cable traps.
The system works as follows: When the MRI scanning is initiated, the Control unit receives an input trigger signal from the MRI scanner which initiate the pre-programmed stimulation sequence (fMRI design) and settings (volt, pulse width and frequency). The Lead unit converts the electric stimulus to an optical signal that is transmitted through wave guides to the MRI unit inside the scanner room. Here, the optical signal is converted back to an electric stimulus, which is then looped back to the Lead unit for QA before transmission of the electric stimulus to the implanted electrode in the pig brain during scan.
System adversity test (dry runs)
To ensure that our device does not generate RF noise, which could cause image artefacts, we employed the RF Noise Spectrum sequence, which can be utilized to locate the source of an external RF interference. The RF Noise Spectrum sequence is comparable to that of the TIM TRIO sequence for RF noise and spike check developed by The Martinos Center for Biological Imaging and involving Massachusetts General Hospital, Harvard and Siemens, as detailed in http://cbs.unix.fas.harvard.edu/science/core-facilities/neuroimaging/facilities/noise_spike.
The RF Noise spectrum sequence does not use any RF transmitter or gradient pulses, but performs simply by opening the MRI receiver during scan. The sequence runs for 30 seconds and makes 50 measurements in 10KHz frequency ranges around the central frequency of the scanner [0 ± 500 KHz], creating a noise summary image of the mean signal intensity of all the frequency bands. The results are showed as a graphical display in the patient browser, or it can be viewed in real time via the inline display. RF noise is detected as sizeable spikes of the particular frequency band in sight.
We conducted noise scans with our stimulator connected to DBS electrodes placed in a phantom, in the following conditions: a) Stimulation ON, b) Stimulation OFF, c) Alternating stimulation ON (15 sec) vs OFF (15 sec), d) Noise (doors open), and e) Control (no device in the scanner room). To eliminate other potential sources for external RF emission, we also ran the test without the coil placed on the electrodes, and with lights turned both on and off during scan.
All conditions were compared to control by visual inspection for spikes and dissimilarities in the RF Noise on the graphical display, but also in the active measurement output given as the maximum single measurements as well as the average mean of all 30 measurements.
Time accuracy, signal stability and functionality of the device
For quality assurance, we designed the device with a back-loop and logging of the electric signal delivered to the electrode to ensure time-accuracy and constancy of stimulation parameters following the signal conversions in the MRI environment. The back-looped signal was displayed on two systems. First, an oscilloscope was used to monitor amplitude, frequency and pulse width of the back-looped electric stimulus. Moreover, we also monitored the back-looped signal using a color-coded screen of green (stimulation ON) and black (stimulation OFF) and used a time-clock to test that the duration of the blocks (ON and OFF) was in accordance with the preprogrammed fMRI paradigm. Finally, we monitored the functioning of the device for unintentional stops during scan, such as reported in previously DBS-fMRI studies(4) using cables led via waveguides.
Animals, housing and anesthesia
All animal experiments were performed in accordance with the European Communities Council Resolves of 22 September 2010 (2010/63/EU) and approved by the Danish Veterinary and Food Administration’s Council for Animal Experimentation (Journal No.1012-15-2934-00156), and is in compliance with the ARRIVE guidelines (www.nc3rs.org.uk/arrive-guidelines).
In this study, we used four female 9-10-week old pigs (crossbred Landrace, Yorkshire and Duroc, acquired from an agricultural herd) weighing 21 ± 1.5 kg (mean ± SD). After arrival to the Department of Experimental Medicine at the University of Copenhagen, the animals were inspected by a veterinarian and allowed to acclimatize for 1 week. The pigs were housed in groups in adjacent pens (3.8–5.4 m2) with 45–70% humidity, 19–23°C air ventilation, cyclical light (12 h), straw bedding and environment enrichment, in terms of various toys. The animals had free access to tap water, were fed a restricted diet twice a day and fasted 16 h before inducing anesthesia on the experimental day.
On the experimental day, fasting animals were premedicated by intramuscular (i.m.) injection of 0.14 mL/ kg Zoletil® 50 Vet (Virbac, Kolding, Denmark) mixture with 6.25 Pt. xylazine (20 mg/mL) + 1.25 Pt. ketamine (100 mg/mL) + 2 Pt. butorphanol (10 mg/mL) + 2 Pt. methadone (10 mg/mL), weighed and intubated. Anesthesia was induced with 0.5-1 mL intravenous bolus injections of 10 mg/ml propofol (Fresenius Kabi AS, Halden, Norway), while maintaining spontaneous breathing during installation of catheters. Each animal had installed a bladder catheter, a peripheral venous catheter in both ears, one percutaneous femoral artery catheter (Arrow International Inc, Reading, PA, USA) and a small incision catheter in both mamillary veins. All incisions were preceded by local anesthesia given as a subcutaneous injection of 10 mL mixture; 1 Pt. 10 mg/ml xylocaine (AstraZeneca A/S, Copenhagen, Denmark) + 1 Pt. 5 mg/ml Bupivacain (Amgros I/S, Copenhagen, Denmark).
After a short trolley walk to the scanner facilities, the animals were connected to a ventilator with 34% O2 and anesthesia was maintained with 1.5–2.5% isoflurane (Scanvet Animal Health A/S, Fredensborg, Denmark) on all fMRI experimental days. On other non-fMRI experimental days, standard anesthetic was infusion with propofol 15 mg/kg/h. The animal was set up with a slow dripping infusion with isotone saline and monitored throughout by visual inspection, blink reflexes, blood pressure, heart rate, respiratory frequency, capnography, oxygen saturation and temperature.
Surgical planning and surgery
An MR compatible stereotaxic system for pigs (NeuroLogic, Aarhus, Denmark) was used for targeting and implantation of the DBS electrodes and microdialysis probes. The system is comprised of a stereotaxic localizer box with 1) two MRI compatible detachable side plates containing a copper sulfate fiducial marking system that defines a Leksel stereotaxic space, and 2) one detachable arch-based frame for isocentric stereotaxy with a lead implantation device (LID) attached to the arch (5,6).
With the pig placed in prone position and the head fixed in the stereotaxic localizer box attached with the fiducial side plates and an adapted radiofrequency coil, structural T1/T2 MR images (Siemens 3 Tesla mMR hybrid PET-MR scanner) were obtained in each animal to visualize the fiducial markings and the subcortical target(7), followed by manual calculation, using Brain Lab®(BrainLAB AG, Germany), of the stereotaxic coordinates for targets sites and trajectories to the STN.
Next, the fiducial side plates were replaced with the arch-based frame followed by exposure of the sagittal and coronal sutures by a midline incision. Bilateral burr holes to the dura were made to target the medial prefrontal cortex (mPFC) according to a previously validated paradigm (8) and to the MRI guided stereotaxic entry point coordinates to the STN. Next, the guide tube with a blunt cannula was inserted to a location 5 mm above the target coordinate of the STN, where the blunt cannula was removed. The quadripolar (contacts labelled 0, 1, 2, and 3) DBS electrode with internal stylet (Model 3389, Medtronic Inc., Minneapolis, MN, USA) was inserted through the guide tube and gently advanced to the target and stabilized with a two-component surgical adhesive (BioGlue, Cryolife International Inc., Kennesaw, GA, USA) before retracting the stylet and guide tube (9–11). The electrode was fixed with BioGlue to the skull and a frontally placed anchor screw, and the procedure was repeated on the contralateral side. Finally, the incision was sutured and the stereotaxic frame detached from the animal, leaving the electrodes externalized.
The animal was returned to the scanner and placed in the prone position, and the DBS electrodes were connected to the fMRI compatible electrical stimulator with cables and electrodes placed in the z-direction of the PET/MRI scanner.
Before continuing the experiments, preliminary confirmation of the correct position of the electrodes was confirmed by a postsurgical structural T1/T2 MRI scan.
Structural Magnetic Resonance Images
The MR images were generated from a 3T mMR Biograph (Siemens AG, München, Germany) whole-body scanner with a radiofrequency coil (four channels, receive-only, phased array coil) adapted to the pig head. Pre -and postoperative structural T1 and T2 weighted MRI scans were obtained for targeting and implantation of the DBS electrode and alignment of the fMRI scans during data processing and analysis. The postoperative MRI served as preliminary confirmation of the electrode placed in the cerebral target before proceeding with the experimental scans.
The protocol of the T1-wighted 3D magnetic MP-RAGE MRI was: frequency direction (FD), A > > P; slice number,240, field of view (FOV), 250 mm; slice thickness (ST), 1 mm; repetition time (TR), 1900 ms; echo time (TE), 2.44 ms; inversion time (TI), 900 ms; flip angle, 9°; base resolution, 256; acquisition time (AT), 5.04 min.
The protocol of the T2-wighted 3D magnetic fast spin echo (FSE) MRI was: FD, A > > P; slice number, 240, FOV, 250 mm; ST, 1.00 mm; TR, 3200 ms; TE, 409 ms; base resolution, 256; AT, 6:26 min
Stimulation parameters, fMRI design and BOLD protocol
The frequency, pulse width and voltage were set at 125 Hz, 500 or 200 us, and 3 V (voltage) in a rectangular shape delivered between two electrode levels (0–1) of the quadripolar Medtronic 3389 electrode (Medtronics Inc).
The fMRI paradigm was a classic block design, with stimulation turned on and off in a total of 5 cycles. After two volumes of discarded acquisitions to allow for scanner equilibrium, 60 volumes of initial scanning at rest (120 seconds) were performed followed by five stimulus/rest blocks. These blocks each consisted of 3 volumes (6 seconds) of electric stimulus (DBS-ON) and 60 volumes (120 seconds) of rest (DBS-OFF) for a total 375 volumes.
The parameters of the gradient-echo, echo-planar imaging sequence sensitive to the blood-oxygen-level-dependent (BOLD) response were: FD, A > > P; slice number, 42, FOV, 192 mm; ST, 3.00 mm; TR, 2150 ms; TE, 26 ms; flip angle, 78°, base resolution, 64; spatial resolution 3x3x3 mm, AT, 12:07 min
Euthanasia and post-mortem handling
After the final scan, the pigs were euthanized immediately with an overdose of sodium pentobarbital (ScanVet Animal Health A/S, Fredensborg, Denmark) and moved for a post-mortem CT-scan (Dual Source Somatom Definition, Siemens, Munich, Germany) of the brain before removal of the brain and electrodes(12). The CT scan was used as final validation of the correct position of the electrodes in target by co-registration to both the structural MRI and a modified pig brain atlas(13).
Data processing and analysis
After image acquisition, the time series data were processed to obtain maps of brain activation using the JIP analysis toolkit (http://www.nmr.mgh.harvard.edu/~jbm/jip/) customized with the modified pig brain atlas(13) to analyze pig fMRI data.
First, the images were pre-processed by a) Converting DICOM format to NIfTy files, b) Alignment of the structural scan to the modified pig-atlas(13), c) Motion correction (only functional scans) and correction to the hemodynamic input function d) Alignment of the functional scans to the aligned postoperative anatomical scan, and e) Register and spatial smoothing (3 mm) of all fMRI data.
Next, we determined if there was a significant activation associated with DBS-ON compared to DBS-OFF in neocortex and in STN. This was based on a generalized linear modelling (GLM) and cross-correlation with five modeled regressors that include Legendre polynomials to describe signal drift and repetitive finite-impulse response regressors to fit the hemodynamic response function. The regional or focal BOLD response was displayed in a time-dependent fashion and averaged in relation to the event across all cycles for each pig.