The authors declare that this study received philanthropic funding from the John S. &#34;Steve&#34; Dunn, Jr. &#38; 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 &#38; 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.

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
<ol>
	<li>Position the subject for optimal access to the top of the skull: place the subject on the MRI table in preparation for the MRI scan. 
	NOTE: Subject information: domestic swine, female, 25 kg, 14 weeks of age.
	<ol>
		<li>Sedate the subject with ketamine (600 mg intramuscularly [IM]) and midazolam (5 mg IM). Administer the analgesics hydromorphone (4 mg IM), carprofen (100 mg per os), and fentanyl (25 &#181;g topical), the antibiotic ceftriaxone (550 mg intravenously [IV]), and NaCl (0.9% IV).</li>
		<li>Intubate the subject. Maintain anesthesia with 2%-3% isoflurane.</li>
	</ol>
	</li>
	<li>Monitor the subject&#39;s vital signs throughout the procedure.
	<ol>
		<li>Mechanically ventilate at 16-19 respirations/min with a ventilator.</li>
	</ol>
	</li>
	<li>Place the subject on the MRI table in preparation for the MRI scans.</li>
	<li>Place the subject in a prone position with the head facing the MRI bore.</li>
	<li>Place a standard MRI four channel flex coil on the head holder.</li>
	<li>Stabilize the subject&#39;s head with the head holder.</li>
	<li>Raise the torso with towels and foam pads. The goal is for the head to fall slightly downward, with the neck flexed and the snout nearly touching the table. This will help to ensure that the stereotactic frame and infusion cannula fit within the bore of the MRI scanner. Anchor the MRI head holder pins on the bilateral zygoma to keep the head affixed to the MRI table.</li>
	<li>Check that the top of the skull is inclined toward the back of the scanner with the neck flexed. This position enables the surgeon to have access to the top of the scalp when the subject enters the MRI.</li>
	<li>Once set, the MRI table is moved into the bore of the scanner until the subject&#39;s head reaches the end of the bore.</li>
</ol>
2. Planning surgical insertion with MRI-assisted visualization guidance
<ol>
	<li>Prepare the area in a sterile fashion, taking care to avoid the prepared material from getting into the subject&#39;s eyes. Place sterile towels around the surgical area. Place a sterile drape with an opening toward the top of the skull that the surgeon can access.</li>
	<li>Place the fiducial planning grid on the subject&#39;s scalp by affixing the adhesive side of the grid over the patient&#39;s head, centered around the location of where the burr hole will be.</li>
	<li>Peel off the top fluid-filled layer of the grid while firmly holding the lower layer in position.</li>
	<li>Perform the MRI scout scan with the grid set in place. The scan often requires intravenous MR contrast agent administration to visualize the vasculature: use a 1 mmol/mL concentration of the contrast agent gadolinium contrast agent for an infusion volume of 2.5 mL. 
	NOTE: The scout scan is a preliminary image taken before the definitive imaging study. The purpose is for the surgeon to ensure that imaging is carried out close to the region of interest, and to define imaging boundaries. The recommended dose at the 1 mmol/mL concentration, as per the manufacturer, for the contrast agent is 0.1 mL per kilogram that the animal weighs.</li>
	<li>Select the precise brain location for cannula insertion in the MR-guidance software.</li>
	<li>Ensure the software allows the visualization of the surgeon&#39;s planned trajectory for cannula placement, based on the target selected. Ensure the software outputs the trajectory visualization and the corresponding entry point. 
	NOTE: For this study, a site in the frontal cortex was selected to target white matter. This is a location where many human gliomas arise and grow22. It is also a preferential site for dissemination along white matter tracts23. 
	NOTE: Consider the surgeon&#39;s decision for an entry point, target, and desired trajectory to minimize pial and sulcal transgressions and avoid blood vessels.</li>
	<li>Adjust the suggested trajectory, including the desired entry and target points, by manually dragging the projected entry and target points in the software to avoid blood vessels and minimize pial and sulcal transgressions. The trajectory can be changed and viewed in three dimensions.</li>
	<li>Once the desired trajectory is identified based on the surgeon&#39;s preference, run the MR-guidance software to find the entry point on the grid.
	<ol>
		<li>Scroll through the planned trajectory on the scan to find the entry point on the scalp. The software specifies the grid coordinates based on the projection of the planned trajectory on the grid.</li>
	</ol>
	</li>
</ol>
3. Securing the stereotactic frame and adjusting the alignment iteratively through software projection
<ol>
	<li>Assemble the stereotactic frame around the desired entry point coordinates on the grid by first securing the base with six bone-anchored screws and four offset screws.</li>
	<li>Secure the six bone-anchored screws to the skull over the grid, through the scalp. The six anchor screws are used to stabilize the stereotactic frame and avoid any movement during drilling.</li>
	<li>Secure the four offset screws located at the base of the tower through the skin, anchored on the skull. They act as a counter force to tighten the center bone screws, by lifting the frame base to the center screws, and to stabilize the base.</li>
	<li>Once the stereotactic frame base is secure, continue with frame assembly.</li>
	<li>Perform the high-resolution T1-weighted MPRAGE MRI scan, an option in the MRI software, with the frame set in place to capture the frame fiducials and confirm the trajectory.</li>
	<li>Confirm the desired projected cannula insertion trajectory with the software, visualizing the MRI scan and planned trajectory.
	<ol>
		<li>Subsequent 2D turbo spin echo MRI scans are taken to confirm alignment of the frame with the subject once the frame is in place. If there is a misalignment between the current frame position and the desired trajectory, the software outputs adjustment parameters. 
		NOTE: The software calculates the radial difference between the projection of the current position of the stereotactic frame and the defined target point. This error is used to calculate the projected error, which is in turn used to calculate the required adjustments to the frame to minimize it.</li>
	</ol>
	</li>
	<li>Perform the pitch-roll and X-Y adjustments by turning the thumb wheels, as indicated by the output adjustment parameters in the software.</li>
	<li>Repeat the software-enabled MRI visualization of the trajectory and perform rotational and translational adjustments (using the thumb wheels) of the targeting cannula as necessary.</li>
	<li>Using the MR-guidance software, measure the thickness of the skull at the desired trajectory and the total distance to the brain. 
	&#8203;NOTE: The software calculates the distance from the top of the frame (screwed to the skull) to the target point to estimate the total length.</li>
</ol>
4. Drilling and inserting the cannula for infusion
<ol>
	<li>Use an iodine scrub before performing the incision to prevent infection.</li>
	<li>Make a 3 cm incision on the scalp, using a scalpel under the stereotactic frame.</li>
	<li>Set up the frame for drill insertion by performing the adjustments prior to creating the access hole.
	<ol>
		<li>Remove and replace the center guide tube with one that fits a 3.4 mm drill bit for drilling.</li>
	</ol>
	</li>
	<li>Ensure that an assistant is present to hold the frame in place while the surgeon drills with a manual drill to add additional stability to the frame.</li>
	<li>Let the surgeon drill with a manual twist drill to create a 3.4 mm diameter burr hole.</li>
	<li>Set up the frame for the second drill insertion to widen the burr hole and avoid bony collisions that may alter the trajectory.
	<ol>
		<li>Set up the drill with the 4.5 mm drill bit; replace the center guide tube with one that fits this bigger drill bit.</li>
		<li>Create a 4.5 mm burr hole.</li>
	</ol>
	</li>
	<li>Perform an MRI scan to ensure the targeting cannula has returned to the planned trajectory, as drilling through the frame can sometimes shift the cannula.</li>
	<li>Pierce the dura with a sharp stylet.</li>
	<li>Insert the pre-primed frame-compatible infusion cannula. Ensure the cannula has a consistent neutral or positive back pressure to limit the introduction of air bubbles. 
	NOTE: The software provides a specified depth to the planned target.</li>
	<li>Measure the depth on the stereotactic frame-compatible infusion cannula and use the cannula-associated depth stop. This depth stop ensures that the cannula reaches the desired location and does not go beyond it. There is also a lock and dock assembly with an additional screw to ensure the cannula stays at the desired depth.</li>
</ol>
5. Monitoring the infusion with repeated MRI scans
<ol>
	<li>Perform an MRI scan to assess the insertion of the cannula to the correct target location in the brain.</li>
	<li>Start the infusion of the desired agent as a co-infusion with a gadolinium-based contrast agent. 
	NOTE: In this experiment, a 1 mM concentration of gadolinium-based contrast agent was used, but this may need to be adjusted based on the application. A total of 300 &#181;L of infusion volume was administered at a rate of 10 &#181;L/ min, although this may be varied as well.</li>
	<li>Perform an MRI scan at regular time intervals to monitor the infusion and volume of distribution of the cannula-inserted agent in the brain, which can be inferred due to the co-infusion of gadolinium. 
	NOTE: A hyperintense area around the cannula tip indicates the presence of the gadolinium-based contrast agent.</li>
	<li>Once infusion ends, stop the pump. 
	NOTE: The infusion rate used in this study was 30 &#181;L/ min, until the 300 &#181;L volume of the cell suspension was completely infused.</li>
	<li>Let the cannula stay in the brain for 5 min following termination of the infusion prior to removing the cannula. 
	NOTE: The infusion cannula is typically left in place for 5 min following the termination of the infusion to reduce backflow21,24.</li>
	<li>Remove the cannula manually through the frame.</li>
	<li>Remove the frame from the head by disassembling it in reverse order from how it was built.</li>
	<li>Close the incision with a running 3-0 or 4-0 monocryl suture.</li>
	<li>Turn off the isoflurane to prepare for recovery.</li>
	<li>Extubate the subject and allow the subject to recover under observation by the veterinary team.</li>
</ol>

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</ol>

SG, EAS, CJK have the following disclosures: Employed by ClearPoint Neuro. 
All other authors declare no conflicts of interest.

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 &#177; 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&#39;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.

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&#39;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 &#181;L 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.

<table><tbody><tr><td>3 Tesla Siemens MAGNETOM Vida</td><td>Siemens Healthineers</td><td></td><td>70 cm wide-bore 3 Tesla whole body MRI scanner</td></tr><tr><td>Four channel flex coil</td><td>Siemens Healthineers</td><td></td><td>Placed ventrally to allow access to the skull&amp;nbsp;</td></tr><tr><td>MR Neuro Patient Drape</td><td>ClearPoint Neuro, Inc</td><td>NGS-PD-05</td><td>MR Neuro Patient Drape, Marker Pen, Track Ball Cover, Cable Cover</td></tr><tr><td>MR Neuro Procedure Drape Tapered - Long</td><td>ClearPoint Neuro, Inc</td><td>NGS-PD-02-L</td><td>MR Neuro Procedure Drape Tapered, Marker Pen, Track Ball Cover</td></tr><tr><td>MR Neuro Procedure Drape Tapered w/Extension - Long</td><td>ClearPoint Neuro, Inc</td><td>NGS-PD-03-L</td><td>MR Neuro Procedure Drape Tapered w/Extension, Marker Pen, Track Ball Cover</td></tr><tr><td>MR Neuro Scanner Bore Drape w/Extension</td><td>ClearPoint Neuro, Inc</td><td>NGS-PD-04</td><td>MR Neuro Scanner Bore Drape w/Extension</td></tr><tr><td>Scalp Mount Base</td><td>ClearPoint Neuro, Inc</td><td>NGS-SM-01</td><td>Scalp Mount Base and centering too</td></tr><tr><td>Skull Mount Base</td><td>ClearPoint Neuro, Inc</td><td>NGS-SK-01</td><td>Skull Mount Base</td></tr><tr><td>SMARTFrame Accessory Kit</td><td>ClearPoint Neuro, Inc</td><td>NGS -AK-01-11</td><td>Stylet, Lancet, Peel-Away Sheath (2), Ruler, Depth Stop (2)</td></tr><tr><td>SMARTFrame Guide Tubes</td><td>ClearPoint Neuro, Inc</td><td>NGS-GT-01</td><td>15 GA Guide Tube, 18 GA Guide Tube and 16GA Guide Tube</td></tr><tr><td>SMARTFrame Guide Tubes .052&amp;rdquo; / 18 ga</td><td>ClearPoint Neuro, Inc</td><td>NGS-GT-02</td><td>.052&amp;rdquo; Guide Tubes that fit 18 ga devices (5)</td></tr><tr><td>SMARTFrame Guide Tubes .060&amp;rdquo; / 17 ga</td><td>ClearPoint Neuro, Inc</td><td>NGS-GT-03</td><td>.060&amp;rdquo; Guide Tubes that fit 17 ga devices (5)</td></tr><tr><td>SMARTFrame Guide Tubes .064&amp;rdquo; / CP Stylet</td><td>ClearPoint Neuro, Inc</td><td>NGS-GT-04</td><td>.064&amp;rdquo; Guide Tubes that fit ClearPoint Stylets (5)</td></tr><tr><td>SMARTFrame Guide Tubes .068&amp;rdquo; / 16 ga</td><td>ClearPoint Neuro, Inc</td><td>NGS-GT-05</td><td>.068&amp;rdquo; Guide Tubes that fit 16 ga devices (5)</td></tr><tr><td>SMARTFrame Guide Tubes .074&amp;rdquo; / 15 ga</td><td>ClearPoint Neuro, Inc</td><td>NGS-GT-06</td><td>.074&amp;rdquo; Guide Tubes that fit 15 ga devices (5)</td></tr><tr><td>SMARTFrame MR Fiducial</td><td>ClearPoint Neuro, Inc</td><td>NGS-BM-05</td><td>MR Fiducials (5)</td></tr><tr><td>SMARTFrame Scalp Mount Rescue Screw &amp;ndash; Long</td><td>ClearPoint Neuro, Inc</td><td>NGS-RS-02</td><td>Short Scalp Mount Rescue Bone Screws (3)</td></tr><tr><td>SMARTFrame Scalp Mount Rescue Screw &amp;ndash; Short</td><td>ClearPoint Neuro, Inc</td><td>NGS-RS-03</td><td>Long Scalp Mount Rescue Bone Screws (3)</td></tr><tr><td>SMARTFrame Skull Mount Rescue Screw</td><td>ClearPoint Neuro, Inc</td><td>NGS-RS-01</td><td>Skull Mount Rescue Bone Screws (3)</td></tr><tr><td>SMARTFrame Thumb Wheel Extension Set.</td><td>ClearPoint Neuro, Inc</td><td>NGS -TE-01</td><td>Light Hand Controller</td></tr><tr><td>SmartFrame XG Device Guide, 2.5 mm</td><td>ClearPoint Neuro, Inc</td><td>NGS-XG-03</td><td>2.5-mm Device Guide</td></tr><tr><td>SmartFrame XG Device Guide, 3.2 mm</td><td>ClearPoint Neuro, Inc</td><td>NGS-XG-04</td><td>3.2-mm Device Guide</td></tr><tr><td>SMARTFrame XG Drill Guide, 4.5 mm</td><td>ClearPoint Neuro, Inc</td><td>NGS-XG-02</td><td>4.5-mm Drill Guide</td></tr><tr><td>SMARTFrame XG Drill Guide, 6.0 mm</td><td>ClearPoint Neuro, Inc</td><td>NGS-XG-05</td><td>6.0-mm Drill Guide</td></tr><tr><td>SMARTFrame XG Exchangeable Device Guides</td><td>ClearPoint Neuro, Inc</td><td>NGS-XG-01</td><td>Device Guide, 3.4-mm, Device Guide, 14 GA</td></tr><tr><td>SMARTFrame XG MRI-Guided Trajectory Frame</td><td>ClearPoint Neuro, Inc</td><td>NGS-SF-02-11</td><td>Stereotactic Frame, Skull Mount Base, Centering Ring, Dock, Standard Device Lock, Large Device Lock, Screwdriver, Roll Lock Screw w/washer</td></tr><tr><td>SMARTFrame XG MRI-Guided Trajectory Frame, 5 Fr</td><td>ClearPoint Neuro, Inc</td><td>NGS-SF-02-11-5</td><td>Stereotactic Frame, Centering Ring, Dock, 5 Fr Device Lock, Large Device Lock, Screwdriver, Roll Lock Screw w/washer</td></tr><tr><td>SMARTFrame XG MRI-Guided Trajectory Frame, 7 Fr</td><td>ClearPoint Neuro, Inc</td><td>NGS-SF-02-11-7</td><td>Stereotactic Frame, Centering Ring, Dock, 7 Fr Device Lock, Large Device Lock, Screwdriver, Roll Lock Screw w/washer</td></tr><tr><td>SMARTGrid MR Planning Grid</td><td>ClearPoint Neuro, Inc</td><td>NGS -SG-01-11</td><td>Marking Grid and Marking Tool</td></tr><tr><td>SMARTTip MR Drill Kit, 4.5-mm</td><td>ClearPoint Neuro, Inc</td><td>NGS-DB-45</td><td>4.5-mm Drill Bit, 3.2-mm Drill Bit, Lancet, Depth Stop, Ruler</td></tr><tr><td>SMARTTwist MR Hand Drill</td><td>ClearPoint Neuro, Inc</td><td>NGS-HD-01</td><td>Hand Drill</td></tr><tr><td>VentiPAC&amp;nbsp;</td><td>SurgiVet</td><td>V727000 &amp;nbsp;&amp;nbsp;</td><td>Mechanical ventilator</td></tr><tr><td>Wharen Centering Guide</td><td>ClearPoint Neuro, Inc</td><td>NGS-CG-01</td><td>Wharen Centering Guide</td></tr></tbody></table>

magnetic resonance-guided stereotaxy for infusions to the pig brain

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.

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&#39;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64079/64079fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64079/64079fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64079/64079fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64079/64079fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64079/64079fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64079/64079fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64079/64079fig04v2.jpg" /> 
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:&#160;(A)&#160;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:&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64079/64079fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain at JoVE.com

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

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 ...

magnetic-resonance-guided-stereotaxy-for-infusions-to-the-pig-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.3K Views.  Houston Methodist Research Institute. Hier breiden we de toepassing van MR-geleide stereotaxie uit naar de varkenshersenen om de distributie van een infuusmiddel af te leveren en te monitoren. De grootte van de varkenshersenen maakt beeldvorming en neurochirurgische interventies mogelijk die translationeel zijn. We gebruiken het varkensmodel voor MR-geleide stereotactische injectie in een 3T MRI-suite.We rapporteren visueel de implementatie van de stereotaxische procedure bij varkens en beschrijven de aanpassingen van de M...

Video: Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Cancer Research

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System

Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity during sonication. Unfortunately, current clinical treatments for glioblastoma (GBM) are lacking, leading to low long-term survival rates among patients. SDT is a promising method for treating GBM in an effective, noninvasive, and tumor-specific manner. Sonosensitizers preferentially enter tumor cells compared to the surrounding brain parenchyma. The application of FUS in the presence of a sonosensitizing agent generates reactive oxidative species resulting in apoptosis. Although this therapy has been shown previously to be effective in preclinical studies, there is a lack of established standardized parameters. Standardized methods are necessary to optimize this therapeutic strategy for preclinical and clinical use. In this paper, we detail the protocol to perform SDT in a preclinical GBM rodent model using magnetic resonance-guided FUS (MRgFUS). MRgFUS is an important feature of this protocol, as it allows for specific targeting of a brain tumor without the need for invasive surgeries (e.g., craniotomy). The benchtop device used here can focus on a specific location in three dimensions by clicking on a target on an MRI image, making target selection a straightforward process. This protocol will provide researchers with a standardized preclinical method for MRgFUS SDT, with the added flexibility to change and optimize parameters for translational research.

Here, we describe a protocol that details how to perform sonodynamic therapy in an in vivo mouse glioblastoma model using magnetic resonance-guided focused ultrasound.

Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

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