Two adult rhesus monkeys (Macaca mulata) were included in the study. Experiments were performed according to National Institutes of Health guidelines and to protocols approved by the Institutional Animal Care and Use Committee at the University of California San Francisco. NHPs were scanned on a Siemen's Magnetom Avanto 1.5T MRI using an array of 2 custom-built receive-only coils positioned on the left and right sides of the head. Each animal received 2 or 3 infusions of 1 mM gadoteridol (Prohance, Bracco Diagnostics) in 1 hemisphere during a first MRI session, and 2 or 3 infusions in the contralateral hemisphere during a second MRI session. NHP recovered for 2–3 weeks between infusion sessions. NHP were monitored daily over the course of the study by trained veterinary nurses.
Infusions were performed in a research magnet shared between human and nonhuman primate use. Due to institutional regulations prohibiting procedures that expose animal blood products in such an area, the surgical placement and removal of the skull-mounted aiming device (SmartFrame; fig. ) did not occur in the inMRI suite, as would occur in patients. Two weeks prior to infusion, NHPs underwent stereotactic placement of skull-mounted, MRI-compatible, threaded plastic adapter plugs (12 mm diameter × 14 mm height) for later attachment of the SmartFrame. After performing bilateral craniectomies, 1 plug was secured to the skull over each hemisphere with dental acrylic. After placement of the adapter plugs, the animals recovered for at least 2 weeks before initiation of inMRI infusion procedures.
Fig. 1 Description of skull-mounted aiming device. a Basic components of the SmartFrame. b SmartFrame mounted on the left skull plug of a NHP, in a similar orientation to that depicted in a. The cannula (arrow) has been inserted through the fluid stem. A = Anterior; (more ...)
The ClearPoint system consists of the SmartFrame, an infusion cannula, and a software system that communicates with both the MRI console and the operating neurosurgeon in the MRI suite. The ClearPoint software allows registration of the anterior commissure (AC) and posterior commissure (PC) from an initial MRI scan, selection of a target for cannula tip placement in AC-PC space and planning of the cannula trajectory. Although the entry point was relatively fixed in the NHP due to use of the adapter plug, in the clinical system the entry point is modifiable in the precraniotomy planning stage as the trajectory is adjusted. The SmartFrame houses an MRI-visible (gadolinium-impregnated) fluid stem and integrated fiducials, which are detected by the software (fig. ). The fluid stem, which also serves as the infusion cannula guide, is aligned to the target trajectory via both ‘pitch and roll’ axes and an X-Y translational stage. This is accomplished using an attached hand controller resting at the opening of the MRI bore, according to directions generated by the software in response to serial T1 MRI sequences, until the fluid stem alignment matches the chosen target trajectory.
Fig. 2 Sagittal screenshot of target trajectory alignment. The T1 MRI-visible fluid stem (arrowhead), which will hold the infusion cannula, has been aligned by translating the SmartFrame around a fixed pivot point (thick arrow) so that the trajectory (thin arrow) (more ...)
Trajectory Planning and Cannula Insertion
On the day of infusion, NHP were sedated with ketamine (Ketaset, 7 mg/kg, intramuscular) and xylazine (Rompun, 3 mg/kg, intramuscular), intubated and placed on inhaled isoflurane (1–3%). The plug adapter was prepared using sterile techniques and the NHP was placed in an MRI-compatible stereotactic frame in the supine position. Vital signs were monitored throughout the procedure, and an MRI-compatible anesthesia machine was used. The SmartFrame was attached by screwing the base onto the adapter plug over 1 hemisphere. The NHP was moved into the bore and a controller was attached to the SmartFrame by inserting guide wires into each of 4 adjustment knobs. This controller allows the surgeon to manually ‘dial-in’ distance changes to align the cannula to the desired trajectory in 4 planes (pitch, roll, anterior-posterior, medial-lateral) as instructed by the ClearPoint software.
First, a high-resolution anatomical MR scan was acquired for target identification and surgical planning. The scan was a 9-min 3D Magnetization Prepared Rapid Gradient Echo (MPRAGE) acquired with near-isotropic voxel dimensions of 0.7 × 0.7 × 1 mm over a 180-mm field of view (FOV) with 128 slices, an echo time (TE) of 3.76 ms, an inversion time (TI) of 1,100 ms, a repetition time (TR) of 2,170 ms, a 15-degree flip angle and a bandwidth of 130 Hz/pixel. The MPRAGE images were then transferred to the ClearPoint system, where the target for cannula tip placement was selected (fig. ). Next, rapid scans were obtained that allowed the ClearPoint software to detect the position and orientation of the SmartFrame fluid stem. First, a 6-second 2D turbo-spin echo was acquired through the distal fluid stem in an orientation perpendicular to the desired trajectory. The scan was acquired at 1 mm in-plane resolution over a 128-mm FOV with a single 10-mm thick slice, a TE of 41 ms, TR of 704 ms, 2 repetitions, an echo train length of 37 and a bandwidth of 400 Hz/pixel. The software used this image to compare the current SmartFrame trajectory to the target trajectory in order to calculate an expected error for tip placement and generate instructions to adjust SmartFrame alignment via the pitch and roll (fig. ). After these adjustments had been made, the scan was reacquired to measure the new expected error and this process was repeated if necessary.
Fig. 3 Sequence for target trajectory alignment using ClearPoint software. a The selected target. b Pitch (orange/horizontal) and roll (blue/vertical) distances required for proper fluid stem alignment. Note that actual instructions for dialing-in these distances (more ...)
When the expected error fell <1.0 mm, the pitch and roll axes on the SmartFrame were locked and a 26-second 2D turbo-spin echo scan was acquired along the sagittal and coronal planes of the guide stem for fine adjustment of the SmartFrame X-Y stage. Seven slices of 1 mm isotropic resolution were acquired over a 180 × 240 mm FOV with a TE of 22 ms, a TR of 500 ms, 2 repetitions, an echo train length of 7 and a bandwidth 250 of Hz/pixel. The ClearPoint software used these images to generate instructions for fine adjustment of the trajectory, achieved by dialing-in distance changes on the SmartFrame X-Y stage. This process was repeated until the software reported an expected error of <0.5 mm, which typically required no more than 2 iterations (fig. ).
The infusion system included a custom-designed, ceramic, fused silica reflux-resistant cannula that was designed in accordance with previously reported principles developed in our laboratory [10
]. The cannula dimensions are shown in figure . For infusions, the cannula was connected to a loading line containing 1 mM
gadoteridol, and the flow was regulated with a trypan-blue-filled, 1-ml syringe mounted onto an MRI-compatible infusion pump (Harvard Bioscience Company). With the aiming device aligned in its final position, the software reported the expected distance from the target to the top of the guide stem, and this distance was measured from the cannula tip and marked on the cannula using a sterile ink marker. A depth stop was then secured at the marked location and the measured insertion distance was verified. The infusion pump was started at 1 μl/min, and after visualizing fluid flow from the cannula tip when held at the height of the bore, the cannula was inserted through the SmartFrame guide stem and into the brain. When the depth stop encountered the top of the guide stem, it was secured with a locking screw.
Custom-designed infusion cannula. The distance between hash marks is 1 mm.
Infusion and Imaging
Following cannula insertion, repeated multiplanar Fast Low Angle Shot (FLASH) images were obtained every 5 min throughout the duration of the infusion. The FLASH images were acquired at an in-plane resolution of 0.7 × 0.7 × 1 mm with 128 slices over the 180-mm FOV at a TE of 4.49 ms, a TR of 17 ms with 2 repetitions and a bandwidth of 160 Hz/pixel. The first scan was acquired with a 4-degree flip angle to produce a proton-density-weighted image for visualization of the cannula tip. All subsequent scans were acquired with a 40-degree flip angle to increase the T1-weighting and highlight the signal enhancement from gadoteridol in the infusate.
Upon visualization of gadoteridol infusion at the cannula tip, the infusion rates were increased from an initial rate of 1 μl/min in a ramping fashion, 0.5 μl/min every 5 min, to reach a maximum of 3 μl/min. The interface between trypan blue and gadoteridol within the loading line was also marked at the start and finish of the infusion in order to verify that the infused volume matched that reported by the pump. Each NHP first received a small volume infusion in the subthalamic area (16–25 μl) to allow calculation of targeting error, followed by a larger volume infusion (187–230 μl) into the ipsilateral thalamus. In 3 of the infusion sessions, NHP received an additional, smaller volume infusion (17–40 μl) into the substantia nigra (n = 1) or hippocampus (n = 2) to generate further data points for target error calculation. In general, the total time under general anesthesia for NHPs who received 4 sequential infusions was approximately 6 h.
Imaging Data Analysis
Images obtained during RCD were transferred to the ClearPoint system for analysis of targeting error. With the target position hidden from view, the location of the cannula tip was manually selected in the ClearPoint console by identifying the center of the gadoteridol signal in the lower one third of the infusion volume on the first scan demonstrating convection following cannula insertion (fig. ). The software then automatically reported the vector distance between the target site and the actual position of the cannula tip. The average target error for all infusions was later calculated and the 95% confidence interval was determined. Spearman's rank-order correlation was used as a nonparametric measure of the statistical dependence between depth to target and target error.
Images obtained during RCD were transferred to a PACS and accessed by Osirix, an open-source DICOM reader and imaging workstation. Distribution volumes and spatial patterns were analyzed using OsiriX software. Regions of interest (ROIs) were manually traced and 3-dimensional volumetric reconstructions were then generated from these ROIs and used to calculate volume of distribution/volume of infusion (Vd/Vi) ratios.
Each NHP was perfused transcardially 7 days following its second infusion session. Brains were harvested, sliced in 6-mm coronal sections in a brain matrix, postfixed in 4% paraformaldehyde/PBS and cryoprotected in 30% sucrose. A sliding microtome was used to cut 40-μm serial sections for histological processing. For hematoxylin and eosin (HE) staining, free-floating sections were rehydrated and stained with hematoxylin for 15 s, washed with tap water and then differentiated in 0.5% glacial acetic acid/70% alcohol followed by staining in bluing solution. After incubation in eosin, the sections were dehydrated in alcohol and xylene. For glial fibrillary acid protein (GFAP) and Iba1 immunohistochemistry, the sections were first washed 3 times in PBS for 5 min each followed by treatment with 1% H2O2 in PBS for 20 min at room temperature. The sections were then incubated in Sniper blocking solution (Biocare Medical) for 30 min at room temperature followed by incubation with primary antibodies [Iba1, diluted 1:1,000 (Biocare Medical); GFAP, diluted 1:15,000 (Chemicon)] in Da Vinci diluent (Biocare Medical) overnight at room temperature. After 3 rinses in PBS for 5 min each at room temperature, the sections were incubated in Mach 2 horseradish peroxidase polymer (Biocare Medical) for 1 h at room temperature, followed by several washes and colorimetric development with 3,30-diaminobenzidine. Immunostained sections were mounted on slides and sealed with Cytoseal (Richard-Allan Scientific).