A Neural Implant Design Toolbox for Nonhuman Primates

Summary

This paper outlines automated processes for nonhuman primate neurosurgical planning based on magnetic resonance imaging (MRI) scans. These techniques use procedural steps in programming and design platforms to support customized implant design for NHPs. The validity of each component can then be confirmed using three-dimensional (3D) printed life-size anatomical models.

Abstract

This paper describes an in-house method of 3D brain and skull modeling from magnetic resonance imaging (MRI) tailored for nonhuman primate (NHP) neurosurgical planning. This automated, computational software-based technique provides an efficient way of extracting brain and skull features from MRI files as opposed to traditional manual extraction techniques using imaging software. Furthermore, the procedure provides a method for visualizing the brain and craniotomized skull together for intuitive, virtual surgical planning. This generates a drastic reduction in time and resources from those required by past work, which relied on iterative 3D printing. The skull modeling process creates a footprint that is exported into modeling software to design custom-fit cranial chambers and headposts for surgical implantation. Custom-fit surgical implants minimize gaps between the implant and the skull that could introduce complications, including infection or decreased stability. By implementing these pre-surgical steps, surgical and experimental complications are reduced. These techniques can be adapted for other surgical processes, facilitating more efficient and effective experimental planning for researchers and, potentially, neurosurgeons.

Introduction

Nonhuman primates (NHPs) are invaluable models for translational medical research because they are evolutionarily and behaviorally similar to humans. NHPs have gained particular importance in neural engineering preclinical studies because their brains are highly relevant models of neural function and dysfunction^{1,2,3,4,5,6,7,8}. Some powerful brain stimulation and recording techniques, such as optogenetics, calcium imaging, and others, are best served with direct access to the brain through cranial windows^{9,10,11,12,13,14,15,16,17,18,19,20,21,22,23}. In NHPs, cranial windows are often achieved with a chamber and an artificial dura to protect the brain and support long-term experimentation^{8,10,12,17,18,24,25,26,27}. Likewise, headposts often accompany chambers to stabilize and align the head during experiments^{14,15,25,26,28,29,30}. The effectiveness of these components is heavily dependent on how well they fit into the skull. A closer fit to the skull promotes bone integration and cranial health by decreasing the likelihood of infection, osteonecrosis, and implant instability³¹. Conventional design methods, such as manually bending the headpost during surgery^25,29 and estimating the skull curvature by fitting circles to coronal and sagittal slices of magnetic resonance (MR) scans^9,12 can introduce complications due to imprecision. Even the most precise of these create 1-2 mm gaps between the implant and the skull, providing space for granulation tissue to accumulate²⁹. These gaps additionally introduce difficulty placing screws in surgery⁹, compromising the stability of the implant. Customized implants have more recently been developed to improve osseointegration and implant longevity^9,29,30,32. Additional costs have accompanied advancements in custom implant design because of the reliance on computational models. The most accurate methods require sophisticated equipment such as computerized tomography (CT) machines in addition to MR Imaging (MRI) machines^30,32,33 and even computer numerical control (CNC) milling machines for developing implant prototypes^25,29,32,34. Gaining access to both MRI and CT, particularly for use with NHPs, may not be feasible for labs in need of custom-fitted implants like cranial chambers and headposts.

As a result, there is a need in the community for inexpensive, accurate, and non-invasive techniques of neurosurgical and experimental planning that facilitate the design and validation of implants prior to use. This paper describes a method of generating virtual 3D brain and skull representations from MR data for craniotomy location planning and the design of custom cranial chambers and headposts that fit the skull. This streamlined procedure provides a standardized design that can benefit experimental outcomes and the welfare of the research animals. Only MRI is required for this modeling because both bone and soft tissue are depicted in MRI. Instead of using a CNC milling machine, models can be 3D printed inexpensively, even when multiple iterations are required. This also allows for the final design to be 3D printed in biocompatible metals such as titanium for implantation. Additionally, we describe the fabrication of an artificial dura, which is placed inside the cranial chamber upon implantation. These components can be validated pre-surgically by fitting all parts onto a life-size, 3D-printed model of the skull and brain.

Protocol

All procedures involving animals were approved by the Institute for Animal Care and Use Committee at the University of Washington. A total of four adult male rhesus macaques (Macaca mulatta) were used in this study. At the time of MRI acquisition, monkey H was 7 years old, monkey L was 6 years old, monkey C was 8.5 years old, and monkey B was 5.5 years old. Monkeys H and L were implanted with custom chronic chambers at 9 years of age.

1. Skull and brain isolation (Figure 1)

1. Acquire a T1 Quick Magnetization Prepared Gradient Echo (MPRAGE) file of the skull and brain using a 3T MRI machine. Use the following parameters for MRI acquisition³⁵: flip angle = 8°, repetition time/echo time = 7.5/3.69 s, matrix size = 432 x 432 x 80, acquisition duration = 103.7 s, Multicoil, slice thickness = 1 mm, number of averages = 1.
2. Download the folder labeled supplemental_code (Supplemental Coding File 1). This folder should contain the following files: brain_extract.m, brain_extraction.m, make_stl_of_array.m³⁶, stl_write.m³⁷.
3. Add the MRI file to the supplemental_code folder. In the computational software, select the supplemental_code folder as the file path and run brain_extract.m.
4. The following steps outline a semi-automated method of skull and brain isolation using MATLAB (Figure 1), which has been aggregated from prior extraction techniques³⁵. The command window will prompt for the parameters needed for brain and skull isolation and craniotomy visualization. After each response is entered into the command window, click enter.
   1. The command window will first prompt for the name of the MPRAGE file. Type in the filename (e.g., MRIFile.dcm) and confirm that the MRI is properly displayed (Figure 1A).
   2. To isolate the skull (Figure 1B - D), follow the detailed steps that are outlined in the command window. Identify a suitable threshold value that separates the skull from surrounding tissue without eliminating skull matter (Supplementary Figure 1A). Confirm a threshold value by pressing (y).
   3. A similar technique is used to isolate the brain (Figure 1E - G). When prompted in the command window, enter a threshold for the brain. Evaluate the figure that pops up and adjust the threshold if necessary. Ensure that the brain is isolated from the skull and surrounding tissue and that no brain tissue is being removed in the process. Confirm a threshold value by pressing (y).
   4. Proceed to the section of interest.

2. Craniotomy location planning (Figure 2)

1. After the brain and skull have been extracted, input the coordinates of the craniotomy. If the coordinates are not known yet, indicate (n) for no, and a figure will be displayed (Supplementary Figure 1B). Determine craniotomy coordinates by choosing a z-frame (coronal plane) and selecting a point on the chosen z-frame for the craniotomy center.
   1. If the coordinates are known, indicate them with respective x (sagittal), y (axial), and z (coronal) values.
2. Input the craniotomy radius in millimeters (e.g., 10 mm) and choose no outer radius.
3. Specify whether a scale bar is needed for the skull and brain images. Scale bars help confirm that the dimensions of the models are correct.
4. Save brain and skull files as STL for 3D printing if desired (Figure 1D, G).
5. Next, a figure with the brain and craniotomized skull will be displayed. This can be used to verify the access to targeted brain areas. The brain is represented in blue, and the skull in light gray (Figure 2B, E).
6. Choose (n) for completing an SLT size reduction, which is a feature that will be used for future steps (see below).
7. Repeat sections 1 and 2 per craniotomy iteration.

3. Cranial chamber design (Figure 3)

1. Prior to starting the chamber design, confirm the location of the craniotomy and the craniotomy radius using the craniotomy location planning procedure.
2. After the skull and brain isolation have been completed, the next step will be to input the finalized coordinates of the center of the craniotomy. Input the x (sagittal), y (axial), and z (coronal) values.
3. The command window will next prompt for entering the inner and outer radii, which determine the area of the skull to work with for chamber design. Choose an inner radius smaller than the actual craniotomy radius (e.g., 5 mm for a craniotomy radius of 10.0 mm) and a second outer radius larger than the planned radius of the chamber skirt (e.g., 26 mm for a chamber skirt that will have a radius of 22 mm). This will provide a ring-shaped skull structure as a foundation for the chamber to be built onto.
   NOTE: For designing a chamber with a craniotomy radius of 10 mm, a 5 mm inner radius was chosen. This provides an accurate representation of the skull at the craniotomy edge while maintaining a small enough circle that the craniotomy center can be easily identified when the skull representation is exported to the design software. An outer radius of 26 mm was extracted for a chamber of radius 22 mm to ensure that extra skull area is available. The dimensions of the chamber were developed with constraints established by the needs of the experiment. Radii used at this step will be determined by the craniotomy size and the size of the chamber skirt, which is dependent on screw sizes and available space on the skull.
4. Indicate whether scale bars are needed for skull and brain images.
5. Save brain and skull files if desired.
6. A figure will pop up with the brain (in blue) and the skull region (in gray) that was selected (Figure 3A). An STL size reduction then needs to be applied on the selected skull region for easier handling of the file in the Computer-Aided Design (CAD) software.
7. Select (y) to begin the STL size reduction. The size reduction will create an STL file with a reduced file size that can be easily imported into CAD software for custom hardware design.
8. Using the figure with the overlayed brain and skull (Figure 3A), use the mouse to select points on the skull surface to be used for the file reduction. Hold the shift key down to place more than one point.
   1. Place points to cover the region of interest, which in this case is the selected skull region. Place the points as close together as possible to ensure a more precise and accurate representation of the skull (Supplementary Figure 2). Some users may prefer to select ~20 critical points and complete the rest of the chamber design as practice prior to selecting all points of interest for the final product.
   2. When selecting points, it is best to place as many points in the selected region as possible. In general, ~200 points represent the skull curvature well. Place more points around the edges of the selected region to emphasize the boundary between the brain and the skull.
      ​NOTE: Avoid clicking the enter button before finishing placing points across the region, as it will cause the code to progress prematurely, and the point selection process will have to be repeated.
9. Press enter when finished placing points on the selected skull. Type the reduced filename into the command window.
10. Import the file into CAD software for custom chamber design. Start by opening CAD software.
11. Click File > Open and select the filename of the STL reduction from the directory.
    1. Before clicking Open, click the Options button, and in the Import as menu, click Surface Body. Click OK and then Open.
12. Once the STL is imported, check for small holes on the surface, indicated by blue lines. If there are holes in the region of the skull that the chamber will cover (Supplementary Figure 3), complete the fixing holes procedure (Section 6) at step 3.19.1.
13. View the skull surface for the chamber in CAD software as in Figure 3B. Ensure the edges of the selected area are visible in the skull representation.
14. Find the outline of the inner circle at the center of the imported surface to locate the center of the craniotomy. Create a plane aligned with the inner circle by clicking Insert > Reference Geometry > Plane. Use three points evenly distributed along the circumference of the inner circle as the reference points for the plane.
15. Create a circle corresponding to the inner circle by clicking on the circle icon in the Sketch tab. Choose the plane from the previous step as the reference plane and identify points along the edge until the circle preview provides an accurate representation of the inner circle outline. Several different combinations of points may have to be tested to find ones that best fit the inner circle.
16. With the circle as a reference, create a point at the middle of the circle by clicking Insert > Reference Geometry > Point and use the Arc Center option. This point represents the center of the craniotomy.
17. As a reference plane for future extrusions, make a second plane parallel to the initial plane and offset by 10 mm. When choosing the direction of offset, ensure the arrow is pointing upwards from the object.
18. Creating inner ring of chamber (Figure 3C)
    1. Make an axis that extends perpendicularly through both the craniotomy plane and the upper plane by clicking Insert > Reference Geometry > Axis, highlighting the Point and Face/Plane option, and using the upper plane and the center point of the craniotomy as references. Make another point at the intersection of this axis and the upper plane.
    2. Select Extrude Boss/Base and the upper plane as the surface from which to extrude. Make a sketch of the inner ring cross section by creating two concentric circles with the point on the upper plane as the center point (e.g., 11.35 mm and 12.25 mm radii). Select Up to Surface in the direction menu and specify the imported surface as the surface to which to extrude.
    3. Copy the imported surface by selecting Insert > Surface > Move/Copy and raise the copied surface to the height of the inner ring and skirt (e.g., 3.5 mm). Use the Translate option in the Move/Copy menu and translate the surface along the axis perpendicular to both planes.
    4. Perform a circular extruded cut from the upper plane to the copied surface. Start by clicking Extruded Cut and selecting the top surface of the inner ring as the starting point for the extruded cut. Complete the extrusion by choosing the copied surface as the endpoint.
    5. Delete the original imported surface using the Insert > Features > Delete/Keep Body tool. With the Hide/Show tool in the View tab, the copied surface can be hidden to view the inner ring and validate its design.
19. Creating chamber skirt (Figure 3D)
    1. Make a second copied surface offset lower than the existing surface by a thickness of the chamber skirt (e.g., -1.5 mm). In the Translate menu, choose the axis perpendicular to the planes as the point of reference and an offset value to create the new surface below the initial one.
       ​NOTE: Depending on the default direction of the offset direction, the offset value may have to be set as negative to go in the correct direction.
       1. If there are holes in the region that the chamber will cover, follow the steps outlined in section 6 (fixing holes) before continuing with the rest of the chamber design procedure.
    2. Perform an extrusion from the upper plane to the lower surface in the shape of the chamber. Start by selecting Extrude Boss/Base and selecting the upper plane as the extrusion plane.
       1. Follow step 6.2 for handling existing extrusions from the fixing hole procedure.
    3. Sketch the shape of the chamber skirt onto this plane. Make the inner circle of the chamber a circle of the same size as the smaller radius of the inner ring (e.g., 11.35 mm), center it around the point on the upper plane, and make the outer boundary of the chamber skirt using a combination of arcs and lines to maximize skirt area. Extrude to the lower of the two surfaces.
       NOTE: If an error arises with the extrusion, it is likely that the sketch is wider than the surface. In this case, decrease the size of the outer skirt boundary.
    4. Extrude cut from the upper plane to the higher of the two copied surfaces in the shape of the chamber outline.
       1. See step 6.2 for additional information on extrusions left over from the fixing holes procedure.
    5. To reveal the chamber skirt and inner ring, delete both remaining copies of the imported surface. The resulting object should appear similar to that in Figure 3D.
    6. During the process of making the STL reduction and importing it, the model of the skull is mirrored. To compensate for this, the resultant skirt needs to be mirrored. In the Features menu, click Mirror and mirror the skirt across the upper plane. Delete the original skirt using the Delete/Keep Body function.
20. Combining the chamber top and skirt (Figure 3E)
    1. Open the chamber top STL file in the software used to design the chamber skirt. Then, insert the chamber skirt as a part by clicking Insert > Part, selecting the custom skirt in the menu, and clicking anywhere on the screen to import the part.
    2. To align the chamber top and skirt, click Insert > Features > Move/Copy. Select the chamber skirt and click the Constraints button at the bottom of the menu. Highlight the inner ring of the skirt and the inner surface of the chamber top as concentric mates (Supplementary Figure 4A).
       1. Confirm that the top of the skirt is aligned with the bottom of the chamber top, and switch mate alignment direction if required.
    3. Use Move/Copy to translate the skirt downward directly below the chamber top. This will require multiple iterations to find the correct distance so that the chamber top does not extend below the chamber skirt and obstruct the skirt (Supplementary Figure 4B, and Supplementary Figure 5).
    4. Rotate the chamber top to align the gaps between tabs so that one is perpendicular and one is parallel to the midline of the brain. Use the Rotate tool and the existing axis in the center of the object as the axis of rotation. Adjust the degrees of rotation until the chamber top and skirt are in the correct orientation relative to one another.
    5. Connect objects together by extruding from the bottom of the chamber top directly downwards towards the skirt. Use Extrude Boss/Base, select the bottom surface of the chamber top, and create a sketch on this surface with the same inner and outer radii as this ring, using the central axis as the center point. Choose Up To Body as the extrusion direction and indicate the chamber skirt.
    6. Perform an extruded cut from the surface of the chamber top that holds the tabs. After selecting that surface as the extrusion plane, sketch a circle with the same inner radius as the inner ring. Exit sketch and perform a Blind extruded cut that surpasses the bottom of the chamber skirt (e.g., 10 mm).
    7. Add twelve screw holes evenly spaced around the chamber skirt. Place the screw holes so that they are spaced evenly but also far enough apart that they are accessible during surgery but close enough to avoid an unnecessarily large chamber footprint.
    8. Use the Hole Wizard tool for screw-hole placement. Choose parameters in the Hole Specification - Type menu. Parameters should align with the screws that will be used during surgical implantation (e.g., Standard: ANSI Metric, Type: Flat Head Screw - ANSI B18.6.7M, Size: M2, Fit: Loose, Minimum Diameter: 3.20 mm, Maximum Diameter: 4.00 mm, Countersink angle: 90 deg, End Condition: Through All).
    9. Click the Positions tab to begin placing holes. To place a hole, hover over a plane on the chamber and right-click. Place all twelve screw holes, ensuring they are evenly placed and accessible.
    10. If obstructions remain inside a screw hole after it has been placed (Supplementary Figure 6A), choose a different plane to place the hole onto or use the following steps to perform an upwards extruded cut through the hole.
        1. Start the upwards extruded cut by creating a plane parallel to the remaining plane but offset downwards by 0.00001 mm so that the plane is directly under the obstruction.
        2. Perform the extruded cut with the plane created in the last step as the reference. Using a combination of arcs and lines, sketch the shape of the area that needs to be removed. Ensure that the sketch contains any part of the plane that is inside the outer radius of the screw hole (Supplementary Figure 6B). Extrude cut 1 mm upwards.
    11. After placing the screw holes, trim the skirt to reduce sharp edges and minimize unnecessary skirt area. Perform an Extruded Cut from the top surface of the chamber down past the chamber skirt (e.g., 30 mm). Make the extrusion in a shape that will smooth any rough edges and trim the outer skirt area.
        1. Additional custom cuts may be necessary to remove all sharp edges and excess skirt. If areas of the skirt cannot be cut using the top surface of the chamber as the reference plane, create an angled plane and create additional extruded cuts using this plane.
    12. See Figure 3F for a final chamber design representation. This design can be 3D printed and placed on a model brain and craniotomized skull if desired (Figure 3G).

4. Headpost design (Figure 4)

1. Note that the finalized craniotomy center location and the maximum skirt area of the chamber will be required for the headpost design.
2. Input the known craniotomy coordinates (x, y, and z values) into the command window.
3. For the headpost design, only one radius is required to represent the area on the skull that is available surrounding the chamber. At this step, enter the maximum radius of the chamber that was designed in the previous section (e.g., 25 mm). Next, indicate that no outer radius is needed.
4. Use the command window to signify whether scale bars are needed to confirm dimensions.
5. Similar to the previous sections, save brain and skull STL files if needed for 3D printing.
   The next figure that is displayed will show the region of the skull that surrounds the chamber for the creation of a headpost footprint. Extract this region using an STL size reduction to be imported into design software.
6. Select (y) to indicate that an STL size reduction is desired. Select points on the figure with the brain (in blue) and the skull (in gray) overlayed together. Ensure that the points are selected as close together as possible and evenly distributed across the gray skull region (Supplementary Figure 7A). For more details on the point selection process, refer to step 3.8.
7. Press enter after completing point selection to cover the gray skull region where the headpost will sit. Indicate a filename for the downloaded reduced file in the command window.
8. Import the reduced file into CAD software to design the custom headpost footprint. Ensure the file is being imported as a Surface Body.
9. After importing the file, check for holes in the surface indicated by blue lines. If there are holes in the general region that the headpost will cover, the fixing holes procedure (section 6) will need to be completed in step 4.11.
10. The first step of the headpost design is to find a plane on the surface that aligns with the axial plane so that when the headpost top and bottom are combined, the headpost top is perpendicular to the skull (Supplementary Figure 7B, C). If a plane that directly aligns with the axial plane cannot be found on the surface of the skull, create a new plane using an existing plane on the surface and rotating it to properly align it. It is helpful to have a physical 3D skull model that can be used for comparison to the virtual skull representation.
    1. This step may have to be modified several times to create a headpost top that is directly perpendicular to the skull. To change the angle of the headpost top with respect to the headpost footprint, modify the plane used in this step. A couple of planes may have to be tested to find one that sits parallel to the axial plane.
11. Use the plane found or created in the previous step to create a parallel plane 3 mm above the surface that will provide a reference for the orientation of the headpost top.
    1. Complete the fixing holes procedure outlined in section 6 with gaps arising in the headpost region.
12. Creating headpost bottom (Figure 4C)
    1. Click Extrude Boss/Base, select the new plane, and create a sketch of the headpost footprint using a combination of arcs and lines. Make headpost legs of similar length and the angles between them congruent (see example in Figure 4A). Use arcs to connect the legs of the headpost to ensure smooth edges around the footprint and extrude the sketch to imported surface.
       ​NOTE: The number of headpost legs will depend on the space available surrounding the chamber. However, the headpost should have a minimum of three legs to ensure proper mechanical stability.
       1. See step 6.2 for instructions on how to draw around the existing extrusions from the fixing hole procedure.
    2. At this point, the bottom surface of the headpost is available for confirming that the surface matches the curvature of the skull. If 3D printing is desired to check the fit, complete the following four steps.
       1. Delete the imported surface body. Mirror the footprint across the plane created in step 4.10. In the Mirror menu, confirm the Merge Solids box is unchecked.
       2. To verify the footprint matches skull curvature, use the Delete/Keep Body to delete the original footprint, leaving only the mirrored version.
       3. 3D print the object as an STL and place it onto the 3D Skull model to physically test if it matches the skull curvature.
       4. To continue with the headpost design, use the Undo arrow at the top of the toolbar to undo the previous two steps (mirroring and deleting). This should restore the original footprint and surface body.
    3. Create a point at the center of the flat surface on the footprint. Create an axis using this point and the upper reference plane.
    4. Click on the Move/Copy tool and create a copy of the imported surface raised to the thickness of the headpost bottom (e.g., 1.35 mm). Use the axis made in this step as the translational reference and verify that the Copy box is checked to prevent the original surface from being modified.
    5. Perform an Extruded Cut from the flat surface of the headpost footprint to the copied (raised) surface. Delete the original surface and its copy. The resulting part can be seen in Figure 4B.
       1. Follow step 6.3 for existing extrusions from the fixing hole procedure.
    6. Create a new plane parallel to the reference plane but translated upwards or downwards to hover at least 1 mm above the headpost bottom. To determine the length of the translation, use the Measure tool in the Evaluate tab. Make a circular extrusion from the new plane to the headpost bottom to create a platform where the base of the headpost top will sit and ensure the platform is centered around the midline of the skull.
    7. Use the Fillet tool in the Features menu to smooth the intersection between the extrusion and the headpost footprint. Test different radii values using the Asymmetric parameter and choose the largest radii values possible.
    8. At this point, verify the placement of the headpost top platform by 3D printing the current version and testing it against a skull model.
    9. Place screw holes along the headpost bottom using the same technique as was used for the chamber screw holes (step 3.20.7). Add a minimum of three screw holes on each headpost leg. Ensure that the center point of each screw hole is at least 5 mm from the center of the next hole, and the edges of each hole are at least 2.5 mm away from the edge of the leg.
       1. To avoid blood vessels that run under the skull and near the midline, confirm that screw holes do not cross the midline and shift them if needed. The product should look similar to the design in Figure 4C.
    10. Mirror the part using the Mirror tool to compensate for the mirroring that occurs during the importing of the skull surface. Use the top of the circular base as the mirror plane.
    11. Delete the original part using the Delete/Keep Body feature so only the mirrored version remains.
13. Combining the headpost top and bottom (Figure 4D)
    1. Import the headpost top as a Part from the Insert menu. After the part has been highlighted in the menu, click anywhere on the screen to add the part.
    2. Using the Move/Copy function, align the headpost top and bottom. Start by specifying the headpost top as the Body to Move. Then, make the following three mates in the Constraints menu:
       1. Ensure that the top surface of the circular headpost platform and the bottom surface of the headpost top mated coincidentally.
       2. Ensure that the outlined edges of the surfaces in the last mate pair mated concentrically.
       3. Mate a line going vertically along the back leg of the headpost and a line running horizontally along the back of the headpost top (the flat side) perpendicularly. Make sure that the curved face of the top is facing forward (anterior) and the flat face is facing closer to the back leg of the headpost (posterior).
       4. Confirm each connection is in the correct direction and switch the mating directions in the menu if needed (see Supplementary  Figure 8 for an example of mates).
          NOTE: The procedure for combining the custom headpost bottom and top uses a generic headpost top that was designed using CAD software. Here, the top part is designed based on the Crist Instrument's headpost. The mating procedure outlined above is specific to these parts and may have to be adjusted if different mating parts are used.
    3. Ensure that the combined headpost top and bottom looks like Figure 4D.
       1. If the headpost top is not properly aligned, modify the reference plane used in step 4.11.

5. Artificial dura fabrication ¹¹ (Figure 5)

1. Obtain the artificial dura mold (Figure 5B).
2. Create the artificial dura silicone mixture by mixing silicone KE1300-T and CAT-1300 in a 10:1 ratio.
3. Pour 1 mL of the mixture onto the top surface of the cylinder in the center of the mold.
4. To prevent air bubbles, place the mold in a vacuum chamber for about 15 min.
5. Add the second layer of the mold, using the posts on either side of the cylinder to guide the alignment of the piece.
6. Pour 3-4 mL of silicone mixture into the mold and place the clear acrylic piece onto the top of the mold (Figure 5A). Use a C-clamp to clamp the mold together.
7. Check for air bubbles in the optical window and remove them with a vacuum chamber as necessary.
8. Cure the resulting structure overnight at room temperature. Leftover air bubbles are removed through the pressure created when the mold gets clamped prior to curing.
9. Disassemble after curing by removing each molding part and carefully removing the silicone dura.

6. Fixing holes procedure

1. Perform the fixing holes procedure if holes have been found on the skull representation (indicated by blue lines in CAD software). Complete the following steps after the lower surfaces (the surfaces that will end the extrusions) have been created. For the chamber, this is following step 3.19. For the headpost, start this procedure after step 4.12 has been completed.
   1. Hide any surfaces or extrusions besides the lower surface so that the lower surface can be visualized independently.
   2. Use Insert > Surface > Planar to create a planar surface on every face that is in contact with the gap, as well as over the gap if applicable. To specify a surface, select every edge as a bounding entity.
   3. Make planar surfaces until each gap is surrounded, including corners of gaps and edges of lines.
   4. Click Insert > Surface > Knit and select every planar surface surrounding the gap. See Supplementary Figure 9A for a visual of the knitted surfaces.
   5. Create a reference axis at each point along the edge of the knitted surface by choosing Point and Face/Plane as the reference type and selecting a point on the edge of the surface and the upper plane. Repeat for every point on the edge of the knitted surface (Supplementary Figure 9B).
   6. Create a point at the intersection of each axis around the knitted surface with the upper reference plane. Choose Intersection as the reference type and select one axis and the upper plane. Ensure a point is created that corresponds to each axis.
   7. Make a sketch that connects each reference point made in the previous step. Choose Up to Surface for the direction and select the knitted surface as the surface to extrude to.
   8. Repeat steps 6.1.2-6.1.7 for all gaps in the region that the chamber or headpost will cover (see Supplementary Figure 9C for the end result of the fixing holes procedure).
2. When performing the extrusion from the upper reference plane to the lowest surface (Step 3.19.2 or Step 4.12.1), ensure that the chamber/headpost outline is drawn around the existing extrusions.
3. Similarly, when performing the extruded cuts from the upper plane to the higher of the two surfaces (step 3.19.4 or Step 4.12.5), perform the main extruded cut separately from the extrusions that resulted from the fixing holes procedure (Supplementary Figure 10A).
   1. For performing extruded cuts from fixing holes, extrude the uppermost surface of the existing extrusions to a plane on the raised surface that provides a smooth top surface for the chamber or headpost (Supplementary Figure 10B). If the extruded cut creates a rigid surface, use a different plane or perform subsequent extrusions.

Results

These components were previously validated using a combination of MRI visualizations and 3D-printed anatomical models. By comparing the automated craniotomy visualization to the 3D printed craniotomy and the MRI at the location of the craniotomy, it is evident that the virtual craniotomy representation accurately reflects the region of the brain that can be accessed with the specified craniotomy location (Figure 2A-F). Additionally, the accuracy of the automated craniotomy visualization was further evaluated by comparing the virtual representation to existing craniotomies from implantation surgeries (Figure 2E,G). The 3D printed model, automated visualization, MRI, and actual craniotomy highlight the same region, showing the major sulci at the same location and with proportional consistency. The process of brain and skull isolation and subsequent craniotomy visualization takes under 15 min to complete, allowing for several locations to be tested in under 1 h.

The efficacy of the brain isolation procedure was confirmed by comparing the virtual craniotomy to the MRI representation of the craniotomy location (Figure 2B,C,E,F). The similarities indicated that the brain isolation procedure has the capability to represent the correct size, location, and shape of anatomical structures on the brain that are being targeted, such as the sulci.

The combined 3D-printed brain and skull were used as an anatomically accurate model to validate the chamber and headpost designs. Prior to investing in titanium parts, the chamber and headpost were 3D printed in plastic. It was confirmed that the implants fit into the skull and that they were not overlapping with one another or obstructing important anatomical markers. The chamber and headpost design process produced components that matched the curvature of the skull (Figure 3G,I, Figure 4E, Figure 6, Figure 7). The artificial dura was also confirmed to fit adjacent to the inner walls of the chamber with a minor gap to account for adjustments made during implantation. Custom chambers were implanted in two macaques. Contrary to previous chamber design methods⁹, every screw that was attempted to be inserted was able to be screwed in. This is due to the drastic reduction of gaps between the chamber and the skull with the custom fit in comparison to the chamber designed from MRI curvature approximations⁹ (Figure 6A-F). One custom-fit chamber has been implanted for over 2 years, and the other a year and a half. With proper maintenance, there has been no screw loss, infection, or stability issues that have arisen due to these implants (Figure 3I).

The custom headpost and chamber design processes prevent the need for manual adjustments during surgery, which could otherwise add hours to the surgery duration. These techniques also decrease the 1-2 mm gaps that result from curvature approximations²⁹, fostering better implant health and improving experimental outcomes. The refinements prevent complications with the implant and extend implant longevity, therefore also improving animal welfare.

Figure 1: Brain and skull isolation. (A) Layered magnetic resonance image (MRI) coronal slices. (B) Layered binary mask from skull thresholding. (C) Layered slices of the isolated skull from an inverted binary mask. (D) Reconstructed 3D skull. (E) Layered binary mask from brain thresholding. (F) Layered MRI slices of isolated brain. (G) Reconstructed 3D brain. Please click here to view a larger version of this figure.

Figure 2: Craniotomy planning. (A) Craniotomy visualization with 3D printed brain and skull model for Monkey B. (B) Craniotomy visualization in computational software for Monkey B. (C) Craniotomy visualization in magnetic resonance (MR) image for Monkey B. (D) Craniotomy visualization with 3D printed brain and skull model for Monkey H. (E) Craniotomy visualization in computational software for Monkey H. (F) Craniotomy visualization in Magnetic Resonance (MR) image for Monkey H. (G) Image of craniotomy in Monkey H. Please click here to view a larger version of this figure.

Figure 3: Chamber implant design. (A) Skull region (gray) used for STL resolution reduction. (B) Skull STL resolution reduction in SOLIDWORKS. (C) Chamber inner ring, highlighted. (D) Chamber Skirt Design in SOLIDWORKS. (E) Connecting chamber skirt and top. (F) Chamber STL in SOLIDWORKS. (G) 3D printed brain, skull, and chamber. (H) Titanium chamber. (I) Implanted chamber in Monkey H. Please click here to view a larger version of this figure.

Figure 4: Headpost design. (A) Headpost bottom outline on skull STL resolution reduction. (B) Custom-fit headpost footprint. (C) Headpost bottom. (D) Headpost design in SOLIDWORKS. (E) 3D printed headpost on the skull. (F) Titanium headpost. Please click here to view a larger version of this figure.

Figure 5: Artificial dura fabrication. (A) Clamping of silicone mixture using mold. (B) Artificial Dura. This figure has been adapted with permission from Griggs et al.¹¹. Please click here to view a larger version of this figure.

Figure 6: Custom-fit versus skull curvature fit chamber. Chamber designed from MRI curvature estimations on skull⁹ from an (A) anterior view, (B) side view, and (C) posterior view. Custom designed chamber from a (D) anterior view, (E) side view, and (F) posterior view. Please click here to view a larger version of this figure.

Figure 7: Chamber, headpost, and artificial dura on overlaid brain and skull Please click here to view a larger version of this figure.

Supplementary Figure 1: Thresholding and craniotomy location planning. (A) Example binary mask with a suitable threshold. (B) Coronal slice on MRI for identifying craniotomy location. Please click here to download this file.

Supplementary Figure 2: Process of STL File Reduction in MATLAB for the chamber design. Please click here to download this file.

Supplementary Figure 3: Visual representation of a hole in the skull STL resolution reduction. Please click here to download this file.

Supplementary Figure 4: Chamber skirt software screenshots. (A) Inner ring of the chamber skirt and the inner surface of the chamber top as concentric mates. (B) Translating chamber skirt downwards. Please click here to download this file.

Supplementary Figure 5: Chamber skirt and chamber top with and without overlap. (A) Under-view example of overlap between the chamber skirt and the chamber top (Modifies the lower surface of the chamber skirt). (B) Example of no overlap between chamber skirt and chamber top. Please click here to download this file.

Supplementary Figure 6: Planes obstructing screw holes and elimination of obstruction. (A) Example of planes obstructing the screw holes following screw hole placement. (B) Outline of extruded cut to eliminate surfaces inside of screw holes. Please click here to download this file.

Supplementary Figure 7: Point selection and the axial plane of the skull. (A)Point selection for headpost design. (B) Upper view of the plane parallel to the axial plane of the skull. (C) Side view of the plane parallel to the axial plane of the skull. Please click here to download this file.

Supplementary Figure 8: Example of mates. (A) First mate - Top surface of the circular headpost platform and the bottom surface of the headpost top as concentric mates. (B) Second mate - Edge of the top surface of circular headpost platform and edge of the bottom surface of the headpost top as concentric mates. (C) Third mate - A line going vertically along the back leg of the headpost and a line running horizontally along the back of the headpost top as perpendicular mates. Please click here to download this file.

Supplementary Figure 9: Fixing holes procedure. (A) Knitted surfaces surrounding the gap in the imported surface. (B) Axis on each point at the edge of the knitted surface. (C) End result of fixing holes procedure. Please click here to download this file.

Supplementary Figure 10: Performing extruded cut. (A) Extruded cut surrounding extrusions from fixing holes procedure. (B) Example extruded cut to a plane on the top surface of the chamber bottom. Please click here to download this file.

Supplemental Coding File 1: Coding files for the protocol. Please click here to download this file.

Discussion

This paper outlines a straightforward and precise method of neurosurgical planning that is not only beneficial for the development of components used for NHP cranial window implantation but also transferrable to other areas of NHP neuroscience research^13,15,25. In comparison to other current methods of NHP implant planning and design^25,29,30, this procedure has the potential to be adopted by more neuroscience labs because it is simple and economical. While CT is commonly used for skull modeling^32,38, this protocol provides sufficient modeling detail for both the brain and the skull using only MRI scans. Existing methods require both MRI and CT scans for brain and skull isolation^30,32,33, while this method eliminates additional costs and challenges of CT imaging. An additional benefit is that this model does not require the alignment of MRI and CT scans, saving significant time and preventing issues associated with poor alignment³⁹. Generating both brain and skull models from a single imaging file produces highly compatible models easily combined for craniotomy visualization. This feature is particularly useful for iterative craniotomy testing processes because rather than combining and aligning files from separate programs^30,33, both models are generated in one software from a single input file and display automatically within seconds. This allows for efficient confirmation of brain and skull modeling accuracy and ensures implants will match the skull curvature in vivo. This also eliminates iterative 3D printing of the skull previously required for determining the optimal craniotomy location³⁵, thus saving tens of hours of printing per iteration. Our software-based technique, by comparison, takes around 10-15 min to generate each craniotomy iteration.

Identifying the implant location relative to the frontal, parietal, and temporal skull regions, as well as other skull features, has immense benefits for surgical and experimental planning. This feature is capitalized on to custom design the headpost footprint with respect to the chamber footprint. For any NHP neuroscience research, this spatial modeling feature can be adapted to design components from anatomical planes, MRI coordinates, anatomical features of the brain and skull, and with respect to existing implants. By doing this, the possibility of unforeseen issues during or after implantation is drastically reduced. This procedure also has the ability to create implants that span multiple brain areas from different planes while maintaining a tight fit to the skull.

The method highlighted here creates a circular chamber and allows for a headpost to be designed around the chamber. However, the procedure here has the potential to accommodate other shapes through the modification of the Chamber Skirt Design section. The same is true for the headpost design – the procedure allows for different numbers of legs and other custom shapes to be created, with the shape being primarily dependent on the available space around the chamber. The shape of the skull STL reduction, which is currently a ring for the chamber design, could be further modified to create different skull STL reduction shapes tailored to the need of particular chamber or headpost designs, facilitating more efficient adaptation.

Although this process effectively creates customized implants, there are steps that can be improved upon for more efficient production. As mentioned before, aligning the top of the headpost perpendicular to the skull is an iterative process with the outlined method in this paper due to the difficulty of identifying the skull orientation in the design software. To streamline the process of positioning the headpost top on the bottom part, additional markers could be placed on the virtual skull representation to indicate axial, sagittal, and coronal planes. The protocol also has the potential to be further automated for increased ease of use. While the skull STL reduction method discussed in this protocol is effective for designing implants, it could be made faster and more consistent with further automation. Our validation procedure requires 3D printing of the skull and implant prototypes for verification that the implants matched the curvature of the skull. This step could potentially be eliminated by creating a method of virtual 3D visualization that combines the brain, skull, chamber, headpost, and artificial dura together.

Our platform provides an entirely virtual process of craniotomy planning and custom implant design. The final designs can be 3D printed and verified on a life-size physical model³⁵. Contrary to existing methods, our protocol does not require costly product iterations or access to expensive machinery like CNC milling machines^29,34. Similar to other existing methods of implant design^{9,12,29,30,32,33,40}, this method completely relies on an imaging modality to accurately depict anatomical structures. Any inaccuracy present in the MRI scan or changes in brain or skull anatomy between MRI and surgery may compromise the efficacy of the implant. Therefore, proper planning for MRI acquisition is essential to optimizing implant design.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D Printing Software (Simplify 3D) (Paid)	Simplify3D	Version 4.1	Used for 3D printing using MakerGear printer
C-Clamp	Bessey	CM22	Used for artificial dura fabrication, 2-1/2 Inch Capacity, 1-3/8 Inch Throat
Formlabs Form 3+ 3D Printer	Formlabs	Form 3+	Used for precise 3D printing
MakerGear M2 3D Printer	MakerGear	M2 revG	Used for 3D printing implant prototypes
MATLAB (Paid)	MathWorks	R2021b	Used for brain and skull isolation, virtual craniotomy visualization and skull STL reduction
Phillips Acheiva MRI System	Philips	4522 991 19391	Used for non-human primate imaging
Photopolymer Resin	Formlabs	FLGPGR04	1L, Grey, used for precise 3D prints with Formlabs printer
PreForm Print Preparation Software	Formlabs	Version 2.17.0	Used for 3D printing with Formlabs printer
Printing Filament (PLA)	MatterHackers	88331	PLA 1.75 mm White. Used for 3D printing with MakerGear printer
Silicone CAT-1300	Shin-Etsu		Used for artificial dura fabrication
Silicone KE1300-T	Shin-Etsu		Used for artificial dura fabrication
SolidWorks (Paid)	Dassault Systems	2020	Used for chamber and headpost design
Syn.Flex-S Multicoil	Philips	45221318123	Used for non-human primate imaging