The protocol presented here demonstrates stereotaxy to the pig brain using convection-enhanced infusions, with real-time magnetic resonance imaging (MRI) visualization guidance and real-time infusion distribution visualization.
The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide precise infusions. The subject was positioned prone in the MR bore for optimal access to the top of the skull with the torso raised, the neck flexed, and the head inclined downward. Two anchor pins anchored on the bilateral zygoma held the head steady using the head holder. A magnetic resonance imaging (MRI) flex-coil was placed rostrally across the head holder so that the skull was accessible for the intervention procedure. A planning grid placed on the scalp was used to determine the appropriate entry point of the cannula. The stereotactic frame was secured and aligned iteratively through software projection until the projected radial error was less than 0.5 mm. A hand drill was used to create a burr hole for insertion of the cannula. A gadolinium-enhanced co-infusion was used to visualize the infusion of a cell suspension. Repeated T1-weighted MRI scans were registered in real time during the agent delivery process to visualize the volume of gadolinium distribution. MRI-guided stereotaxy allows for precise and controlled infusion into the pig brain, with concurrent monitoring of cannula insertion accuracy and determination of the agent volume of distribution.
In this protocol, we describe the application of an interventional magnetic resonance imaging (iMRI) stereotactic system for cannula placement and real-time visualization of infusions into the pig brain. The development of iMRI systems allows for accurate catheter placement1. iMRI allows for visualization of the distribution of the infusion agent in the brain of patients under general anesthesia1,2 to evaluate the accuracy of the procedure in real time.
The MR-guided stereotactic system is a targeted platform that allows for sub-millimeter targeting accuracy1. It uses a skull-mounted aiming device in conjunction with dedicated software that provides anatomical imaging of the brain with projected lead insertion trajectories and adjustment parameters. iMRI guidance for stereotactic surgical intervention to the brain has proven effective in clinical applications, such as deep brain stimulation in the treatment of Parkinson's disease2,3,4,5, focal ablation for the treatment of epilepsy6,7, and convection-enhanced delivery (CED) of drugs to the central nervous system8,9.
The CED method is used to directly deliver therapeutic agents to the central nervous system using fluid convection. This is based on a small hydrostatic pressure gradient that enables the flow of an infusate from the tip of the infusion cannula into the surrounding extracellular space10. Stereotactic methods are used to deliver high concentrations of macromolecules, small molecules11,12, cell transplantation13,14,15, or therapeutic agents into the chosen brain tissue target, circumventing the blood-brain barrier. Factors such as permeability, diffusion coefficients, back pressure, uptake, and clearance mechanisms affect the diffusion of the therapeutic agents16. This technique utilizes a gadolinium-based co-infusate1 for clinical CED, to monitor the infusion agent in real time into the parenchymal target. Parameters such as the volume of distribution in the tissue and related kinetics following targeted accuracy are monitored with iMRI.
CED studies of infusion agents via an MR-guided stereotaxy system have been studied in non-human primates, resulting in accurate, predictable, and safe procedures. Infusion cannula placement accuracy has been shown to reach sub-millimeter placement error17. The system provides a predictable infusion distribution, with an observed linear increase in distribution volume with infusion volume, leading to a subsequently introduced reflux-resistant cannula for CED infusions18. This iMRI infusion procedure was reported to incur no untoward effects in non-human primates19.
Here, we expand the application of MR-guided sterotaxy to the pig brain, to deliver and monitor the distribution of an infusion agent consisting of a 300 µL cell suspension. The size of the pig brain allows for imaging and neurosurgical interventions that can be applied clinically to humans, which is not possible in smaller animal models of disease20. Furthermore, the immune system of the pig produces similar responses to that of humans in terms of responses to biological or other therapeutic agents21. Therefore, working with this animal species for stereotactic drug delivery procedures has direct translational clinical implications and may be logistically easier than with non-human primate research.
We used a pig model (domestic swine, female, 25 kg, 14 weeks of age) for MR-guided stereotaxy. The visual implementation of the stereotactic procedure in pigs is reported in this study. We describe the adaptations of the space to accommodate a pig head, visualization of the procedure both in video and images, and concurrent MR imaging to evaluate infusate distribution in the pig brain. MR-guided stereotaxy was performed in a 3T MRI space.
With this experiment, our group demonstrates the performance of MR-guided stereotaxy in the pig brain, and a basic imaging timeline to track infusions within the brain. The general technique for clinical stereotaxy performed in humans can be applied to the swine skull and brain.
The overall goal of this procedure is to perform MR-guided stereotaxy in the pig brain with real-time MRI visualization guidance. This is achieved by first positioning the subject prone in the MRI bore for optimal access to the top of the skull. The second step is to plan the surgical insertion with MRI-assisted visualization guidance, which involves the placement and scan of a fiducial grid to determine the appropriate entry point for a pre-planned trajectory. This is achieved with a high-resolution (1 mm isotropic) T1-weighted 3D magnetization prepared rapid gradient echo (MPRAGE) scan, in a duration of 7 min and 44 s. Next, we secure the stereotactic frame on the head, and adjust the alignment iteratively through software projection until the projected radial error is less than 0.5 mm. Fast 2D turbo spin echo scans (duration of 13 s) in oblique orientations provide image guidance. Then, an incision is made on the skin, and a hand drill is used to create a burr hole for insertion of the infusion cannula at the predefined coordinates. The final step is to monitor the infusion with repeated T1-weighted MRI scans (3D MPRAGE; 1 min 45 s) in real time with gladolinium co-infusion. The results show that MR-guided stereotaxy allows for precise and controlled infusion into the pig brain, based on real-time MR guidance and subsequent T1-weighted 3D MPRAGE MRI scans (1 mm isotropic resolution) used to visualize the volume of distribution.
The study was approved by the Institutional Animal Care and Use Committee at Houston Methodist Research Institute, IACUC approval number IS00006378. All experimental methods were performed in accordance with the relevant national and institutional guidelines and regulations.
1. Animal positioning
2. Planning surgical insertion with MRI-assisted visualization guidance
3. Securing the stereotactic frame and adjusting the alignment iteratively through software projection
4. Drilling and inserting the cannula for infusion
5. Monitoring the infusion with repeated MRI scans
The pig position in the MRI scanner provides optimal access for the surgeon to operate and clearance for the stereotactic frame and infusion cannula (Figure 1). The subject's torso was raised with towels and foam pads. This enabled the head to fall slightly downward at the end of the MR bore, and therefore ensured that the stereotactic frame and infusion cannula insertion location were optimally accessible for the surgeon.
The MRI-guided visualization allows for precise planning and insertion of a cannula to the brain (Figure 2). The MR-guidance software provides the insertion point to achieve the desired trajectory.
The stereotactic frame was scanned in the software, and it was adjusted to effectively reach the desired location (Figure 3). In this demonstration, a location in the frontal cortex was chosen. Once the frame was set, the software was used to estimate the thickness of the pig skull, the distance to the desired location from the frame base, and the frame parameter adjustments to reach the desired location. In this case, for the location and insertion angle selected, the thickness of the skull that the cannula would traverse was 4.7 mm, and 4.4 mm from the interior surface of the skull to the surface of the brain (Figure 3A).
Finally, iterative interoperative MRI scans after the cannula infusion showed how the infusion was delivered to the brain tissue (Figure 4). These scans also provided a comparison of the cannula projection (blue rectangle) and projected cannula trajectory (yellow rectangle), which show the effectiveness of this technique in reaching the desired location. MR scans were taken at regular intervals of 4-6 min and finalized with 10 and 30 min scans. The gadolinium-enhanced infusion was tractable in these scans, which provided a real-time visualization of the volume of distribution of the agent.
Figure 1: Subject position on the MRI table. The torso is raised, the neck flexed, and the head inclined downward. (A) Before entering the MR bore. (B) Subject positioned through the MR bore for optimal access to the top of the skull. Please click here to view a larger version of this figure.
Figure 2: MR-guided stereotaxy visualization. (A) Visualization of the planned trajectory. The software outputs the entry point location in the grid, placed on the scalp. (B) Entry point location on the scalp. Please click here to view a larger version of this figure.
Figure 3: Intervention trajectory after the frame is secured on the skull. (A) Measurements of bone depth and distance to the brain. (B) Stereotactic frame on the skull, with a burr hole created with a hand drill. (C) Stereotactic frame and 3D reconstruction projection on the software. Please click here to view a larger version of this figure.
Figure 4: Time-lapse of the gadolinium-enhanced infusion agent. The hyperintense area around the cannula tip indicates the presence of gadolinium. Repetitive MR scans were acquired across time to track the volume of distribution of the agent during infusion: (A) t = 0, (B) t = 4 min, (C) t = 8 min, (D) t = 12 min, (E) t = 20 min, (F) t = 26 min; and after the infusion ended: (G) t = 36 min, and (H) t = 60 min. Visualization of the co-infused agent occurs after 4 mins. The blue rectangle is the measured-cannula placement, while the yellow rectangle shows the projected cannula trajectory. Please click here to view a larger version of this figure.
This protocol presents the performance of MR-guided stereotaxy to the pig brain inside a 3T MR machine with the possibility of sub-millimeter targeting accuracy, as achieved in previous studies1,4,17,18,25. Previous cadaver experiments with MR-guided stereotaxy showed a radial error of 0.2 ± 0.1 mm1. In this report, the final depth error with respect to the planned trajectory was 1.4 mm due to online evaluation and adjustment of the trajectory by the surgeons. The final depth error was comparable to radial error findings (under 2 mm) for clinical implementations of iMRI stereotactic procedures in humans26.
Here, we demonstrate the placement of the subject on the MRI table, with its trunk lifted such that the head can fall slightly downward and point outward toward the end of the MR bore. This head placement is critical to provide the surgeon with space to perform the procedure. The stereotactic frame allows for precise and controlled infusion into pig brain models. Additionally, the real-time MR imaging allows for accurate determination of the volume of distribution. Pigs, as large animal models for infusions tracked in real time in MRI, present the possibility of the study of drug delivery to the brain, cell delivery, and other agents of translational value.
The pig has distinct anatomical differences to consider, compared to humans or non-human primates. As pigs grow, the size of the body in the MR bore becomes a challenge. The shape of the head and torso are different from humans, which proves challenging to accommodate for optimal access to the brain for the surgeon, both for the surgical procedure and cannula insertion in the space outside the MR bore. Therefore, it is critical to position the subject in a way that the surgeon has access to the head from the end of the MR bore.
The difference in skull thickness between pigs and humans is a factor to consider. In this protocol, the iMRI visualization allowed for precise estimation of the skull thickness for an efficient burr hole procedure. Given the use of these minimally invasive neurosurgical tools, animal recovery was uneventful.
The MR-guided visualization provides real-time guidance for access to the pig brain, cannula insertion, and monitoring of the infusion agent. The drilling process, tissue deformation, and/or disruption of white matter tracts have been reported to contribute to difficulties in agent delivery to the brain25. Iterative MR scans during the planning and cannula insertion provide the capability for small adjustments. Additionally, infusion parameters such as the rate of infusion or accuracy of the cannula insertion could be changed in real-time or paused, as dictated by the intra-procedural imaging. Finally, an appropriate balance of the gadolinium-based co-infusate must be selected, to obtain a clear evaluation of the volume of distribution of the agent.
The over-concentration of the gadolinium-based contrast agent may have obscured its distribution in the MRI scans27, showing a black spot around the cannula tip, surrounded by a hyperintense area that showed the outer limits of the infusion volume. Available footage of the procedure is limited due to the constraints associated with filming in the limited MRI space around the surgeon's work area. The intraoperative video footage was used to guide the protocol description.
Infusion agents via MR-guided stereotaxy in pigs and other large animal models has resulted in accurate, predictable, and safe procedures. Demonstrating iMRI stereotaxy in pigs provides the basis for the scalability of research treatments that high hold translational value to humans. Pig models have been widely used to study immunological responses due to their similarity to the human response compared to other species28. Therapeutic agents delivered to the brain can be studied in the context of precise target infusion, with the added benefit of real-time MRI visualization of the infusion location, necessary adjustments, and intra-operative evaluation of its distribution in the tissue.
The authors declare that this study received philanthropic funding from the John S. "Steve" Dunn, Jr. & Dagmar Dunn Pickens Gipe Chair in Brain Tumor Research at Houston Methodist. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.
This work was funded in part by grant number RP190587 from the Cancer Prevention and Research Initiative (CPRIT) and the Houston Methodist Foundation.
The authors thank Vi Phan and Lien My Phan, from the Translational Imaging Center at Houston Methodist Research Institute, for their assistance with MR imaging.
The authors declare that this study received philanthropic funding from Paula and Rusty Walter and Walter Oil & Gas Corp Endowment at Houston Methodist. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.
Name | Company | Catalog Number | Comments |
3 Tesla Siemens MAGNETOM Vida | Siemens Healthineers | 70 cm wide-bore 3 Tesla whole body MRI scanner | |
Four channel flex coil | Siemens Healthineers | Placed ventrally to allow access to the skull | |
MR Neuro Patient Drape | ClearPoint Neuro, Inc | NGS-PD-05 | MR Neuro Patient Drape, Marker Pen, Track Ball Cover, Cable Cover |
MR Neuro Procedure Drape Tapered - Long | ClearPoint Neuro, Inc | NGS-PD-02-L | MR Neuro Procedure Drape Tapered, Marker Pen, Track Ball Cover |
MR Neuro Procedure Drape Tapered w/Extension - Long | ClearPoint Neuro, Inc | NGS-PD-03-L | MR Neuro Procedure Drape Tapered w/Extension, Marker Pen, Track Ball Cover |
MR Neuro Scanner Bore Drape w/Extension | ClearPoint Neuro, Inc | NGS-PD-04 | MR Neuro Scanner Bore Drape w/Extension |
Scalp Mount Base | ClearPoint Neuro, Inc | NGS-SM-01 | Scalp Mount Base and centering too |
Skull Mount Base | ClearPoint Neuro, Inc | NGS-SK-01 | Skull Mount Base |
SMARTFrame Accessory Kit | ClearPoint Neuro, Inc | NGS -AK-01-11 | Stylet, Lancet, Peel-Away Sheath (2), Ruler, Depth Stop (2) |
SMARTFrame Guide Tubes | ClearPoint Neuro, Inc | NGS-GT-01 | 15 GA Guide Tube, 18 GA Guide Tube and 16GA Guide Tube |
SMARTFrame Guide Tubes .052” / 18 ga | ClearPoint Neuro, Inc | NGS-GT-02 | .052” Guide Tubes that fit 18 ga devices (5) |
SMARTFrame Guide Tubes .060” / 17 ga | ClearPoint Neuro, Inc | NGS-GT-03 | .060” Guide Tubes that fit 17 ga devices (5) |
SMARTFrame Guide Tubes .064” / CP Stylet | ClearPoint Neuro, Inc | NGS-GT-04 | .064” Guide Tubes that fit ClearPoint Stylets (5) |
SMARTFrame Guide Tubes .068” / 16 ga | ClearPoint Neuro, Inc | NGS-GT-05 | .068” Guide Tubes that fit 16 ga devices (5) |
SMARTFrame Guide Tubes .074” / 15 ga | ClearPoint Neuro, Inc | NGS-GT-06 | .074” Guide Tubes that fit 15 ga devices (5) |
SMARTFrame MR Fiducial | ClearPoint Neuro, Inc | NGS-BM-05 | MR Fiducials (5) |
SMARTFrame Scalp Mount Rescue Screw – Long | ClearPoint Neuro, Inc | NGS-RS-02 | Short Scalp Mount Rescue Bone Screws (3) |
SMARTFrame Scalp Mount Rescue Screw – Short | ClearPoint Neuro, Inc | NGS-RS-03 | Long Scalp Mount Rescue Bone Screws (3) |
SMARTFrame Skull Mount Rescue Screw | ClearPoint Neuro, Inc | NGS-RS-01 | Skull Mount Rescue Bone Screws (3) |
SMARTFrame Thumb Wheel Extension Set. | ClearPoint Neuro, Inc | NGS -TE-01 | Light Hand Controller |
SmartFrame XG Device Guide, 2.5 mm | ClearPoint Neuro, Inc | NGS-XG-03 | 2.5-mm Device Guide |
SmartFrame XG Device Guide, 3.2 mm | ClearPoint Neuro, Inc | NGS-XG-04 | 3.2-mm Device Guide |
SMARTFrame XG Drill Guide, 4.5 mm | ClearPoint Neuro, Inc | NGS-XG-02 | 4.5-mm Drill Guide |
SMARTFrame XG Drill Guide, 6.0 mm | ClearPoint Neuro, Inc | NGS-XG-05 | 6.0-mm Drill Guide |
SMARTFrame XG Exchangeable Device Guides | ClearPoint Neuro, Inc | NGS-XG-01 | Device Guide, 3.4-mm, Device Guide, 14 GA |
SMARTFrame XG MRI-Guided Trajectory Frame | ClearPoint Neuro, Inc | NGS-SF-02-11 | Stereotactic Frame, Skull Mount Base, Centering Ring, Dock, Standard Device Lock, Large Device Lock, Screwdriver, Roll Lock Screw w/washer |
SMARTFrame XG MRI-Guided Trajectory Frame, 5 Fr | ClearPoint Neuro, Inc | NGS-SF-02-11-5 | Stereotactic Frame, Centering Ring, Dock, 5 Fr Device Lock, Large Device Lock, Screwdriver, Roll Lock Screw w/washer |
SMARTFrame XG MRI-Guided Trajectory Frame, 7 Fr | ClearPoint Neuro, Inc | NGS-SF-02-11-7 | Stereotactic Frame, Centering Ring, Dock, 7 Fr Device Lock, Large Device Lock, Screwdriver, Roll Lock Screw w/washer |
SMARTGrid MR Planning Grid | ClearPoint Neuro, Inc | NGS -SG-01-11 | Marking Grid and Marking Tool |
SMARTTip MR Drill Kit, 4.5-mm | ClearPoint Neuro, Inc | NGS-DB-45 | 4.5-mm Drill Bit, 3.2-mm Drill Bit, Lancet, Depth Stop, Ruler |
SMARTTwist MR Hand Drill | ClearPoint Neuro, Inc | NGS-HD-01 | Hand Drill |
VentiPAC | SurgiVet | V727000 | Mechanical ventilator |
Wharen Centering Guide | ClearPoint Neuro, Inc | NGS-CG-01 | Wharen Centering Guide |
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