This project was supported by the Eunice Kennedy Shiver National Institute of Child Health &#38; Human Development of the National Institutes of Health under Award Number K12HD073945, the Washington National Primate Research Center (WaNPCR, P51 OD010425), the Center for Neurotechnology (CNT, a National Science Foundation Engineering Research Center under Grant EEC-1028725) and University of Washington Royalty Research Funds. Funding to the Macknik and Martinez-Conde labs for this project came from a BRAIN Initiative NSF-NCS Award 1734887, as well as NSF Awards 1523614 &#38; 1829474, and SUNY Empire Innovator Scholarships to each professor. We thank Karam Khateeb for his help with agarose preparation, and Toni J Huan for technical help.

All animal procedures were approved by the University of Washington Institute for Animal Care and Use Committee. Two male rhesus macaques (monkey H: 14.9 kg and 7 years old, monkey L: 14.8 kg and 6 years old) were used.
1. Image acquisition
<ol>
	<li>Transport the monkey to a 3T MRI scanner and place the animal in an MR-compatible stereotaxic frame (Table of Materials).</li>
	<li>Record the standard T1 (flip angle = 8&#176;, repetition time/echo time = 7.5/3.69 s, matrix siz...

<ol>
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This article describes a toolbox for preparation for neurosurgeries in NHPs using physical and CAD models of skull and brain anatomy extracted from MR scans.
While the extracted and 3D printed skull and brain models were designed specifically for the preparation of craniotomy surgeries and head post implantations, the methodology lends itself to several other applications. As described before, the physical model of the skull allows for pre-bending of the head post before surgery, which creates...

Primate research has been a pivotal step in the progression of medical research from animal models to human trials1,2. This is especially so in the study of neuroscience and neural engineering as there is a large physiological and anatomical discrepancy between rodent brains and those of nonhuman primates (NHP)1,2,3. With emerging genetic technologies such as chemogenetics, optogenetics, and calcium imaging that require genetic modification of neurons, neural engineering research studying neural function in NHP&#8217;s has gained special attention as a preclinical model for understanding brain function2,4,5,6,7,8,9,10,11,12,13,14,15,16. In most NHP neuroscience experiments, neurosurgical measures are required for the implantation of various devices such as head posts, stimulation and recording chambers, electrode arrays and optical windows4,5,6,7,10,11,13,14,15,17,18.
Current NHP labs use a variety of methods that often include ineffective practices including sedating the animal to fit the legs of a head post and approximate the curvature of the skull around the craniotomy site. Other labs fit the head post to the skull in surgery or employ more advanced methods of gaining the necessary measurements for implantation like analyzing an NHP brain atlas and magnetic resonance (MR) scans to try to estimate skull curvatures2,10,11,16. Neurosurgeries in NHPs also involve fluid injections, and labs often have no way to visualize the projected injection location within the brain2,4,5,13,14 relying solely on stereotaxic measurements and comparison to MR scans. These methods have a degree of unavoidable uncertainty from being unable to test the physical compatibility of all the complex components of the implant.
Therefore, there is a need for an accurate noninvasive method for neurosurgical planning in NHPs. Here, we present a protocol and methodology for the preparation of implantation and injection surgeries in these animals. The whole process stems from MRI scans, where the brain and skull are extracted from the data to create three dimensional (3D) models that can then be 3D printed. The skull and brain models can be combined to prepare for craniotomy surgeries as well as head posts with an increased level of accuracy. The brain model can also be used to create a mold for the casting of an anatomically accurate gel model of the brain. The gel brain alone and in combination with an extracted skull can be used to prepare for a variety of injection surgeries. Below we will describe each of the steps required for the MRI based toolbox for neurosurgical preparation.

<table>
	<tbody>
		<tr>
			<td>3D Printing Software (GrabCAD Print)</td>
			<td>Stratasys</td>
			<td>Version 1.36</td>
			<td>Used for High quality 3D printing</td>
		</tr>
		<tr>
			<td>3D Printing Software (Simplify 3D)</td>
			<td>Simplify3D</td>
			<td>Version 4.1</td>
			<td>Used for PLA 3D printing</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Benchmark Scientific</td>
			<td>A1700</td>
			<td>Used for making gel brains</td>
		</tr>
		<tr>
			<td>Black Nail Polish</td>
			<td>L.A. Colors</td>
			<td>CNP637</td>
			<td>Used for gel molding</td>
		</tr>
		<tr>
			<td>Cannula (ID 320 um, OD 432 um)</td>
			<td>Polymicro Technologies</td>
			<td>1068150627</td>
			<td>Used to inject dye into gel brain</td>
		</tr>
		<tr>
			<td>Cannula (ID 450 um, OD 666 um)</td>
			<td>Polymicro Technologies</td>
			<td>1068150625</td>
			<td>Used to inject dye into gel brain</td>
		</tr>
		<tr>
			<td>Catheter Connector</td>
			<td>B Braun</td>
			<td>PCC2000</td>
			<td>Perifix for 20-24 Gage epidural catheters; Units per Cs 50</td>
		</tr>
		<tr>
			<td>Dremel 3D Digilab 3D45 printer</td>
			<td>Dremel</td>
			<td>F0133D45AA</td>
			<td>Used for prototyping in PLA</td>
		</tr>
		<tr>
			<td>ECOWORKS</td>
			<td>Stratasys</td>
			<td>300-00104</td>
			<td>Used to dissolve QSR support structures</td>
		</tr>
		<tr>
			<td>Erlymeyer flask</td>
			<td>Pyrex</td>
			<td>4980</td>
			<td>Used for gel molding</td>
		</tr>
		<tr>
			<td>Ethyl cyanoacrylate</td>
			<td>The Original Super Glue Corp.</td>
			<td>15187</td>
			<td>Used to make combined cannula</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td></td>
			<td>3023</td>
			<td>Used for gel molding</td>
		</tr>
		<tr>
			<td>HATCHBOX PLA 3D Printer Filament</td>
			<td>HATCHBOX</td>
			<td>3DPLA-1KG1.75-RED/3DPLA-1KG1.75-BLACK</td>
			<td>1kg Spool, 1.75mm, Red/Black</td>
		</tr>
		<tr>
			<td>Locust Bean Gum</td>
			<td>Modernist Pantry</td>
			<td>1018</td>
			<td>Gumming agent for gel brain mixtures</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2019b</td>
			<td>Used for skull extraction</td>
		</tr>
		<tr>
			<td>McCormick Yellow Food Color</td>
			<td>McCormick</td>
			<td></td>
			<td>Used for dye injection</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Panasonic</td>
			<td>NN-SD975S</td>
			<td>Used for agarose curing</td>
		</tr>
		<tr>
			<td>MR Imaging Software (3D Slicer)</td>
			<td>3D Slicer</td>
			<td>Version 4.10.2</td>
			<td>Used for 3D model generation</td>
		</tr>
		<tr>
			<td>MR Imaging Software (Mango with BET plugin)</td>
			<td>Reasearch Imaging Institute</td>
			<td>Version 4.1</td>
			<td>Used for brain extraction</td>
		</tr>
		<tr>
			<td>Philips Acheiva MRI System</td>
			<td>Philips</td>
			<td>4522 991 19391</td>
			<td>Used to image non-human primates</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td>10X diluted with DI water to 1X</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>WPI</td>
			<td>UMP3T-1</td>
			<td>Used for dye injection</td>
		</tr>
		<tr>
			<td>Pump driver</td>
			<td>WPI</td>
			<td>UMP3T-1</td>
			<td>Used for dye injection</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>General Electric</td>
			<td></td>
			<td>Used to preserve agarose gel</td>
		</tr>
		<tr>
			<td>Scientific Spatula</td>
			<td>VWR</td>
			<td>82027-494</td>
			<td>Used to extract gel molds</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault Systemes</td>
			<td>2019</td>
			<td></td>
		</tr>
		<tr>
			<td>Stratasys ABS-M30 filament</td>
			<td>Stratasys</td>
			<td>333-60304</td>
			<td>Used for high quality 3D printing</td>
		</tr>
		<tr>
			<td>Stratasys F170 3D printer</td>
			<td>Stratasys</td>
			<td>123-10000</td>
			<td>Used for high quality 3D printing</td>
		</tr>
		<tr>
			<td>Stratasys QSR support</td>
			<td>Stratasys</td>
			<td>333-63500</td>
			<td>Used to create supports with ABS model</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>SGE</td>
			<td>SGE250TLL</td>
			<td>Used for dye injection</td>
		</tr>
	</tbody>
</table>

a mri-based toolbox for neurosurgical planning in nonhuman primates

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using data extracted from magnetic resonance imaging (MRI). This protocol allows for the generation of 3D printed anatomically accurate physical models of the brain and skull, as well as an agarose gel model of the brain modeling some of the mechanical properties of the brain. These models can be extracted from MRI using brain extraction software for the model of the brain, and custom code for the model of the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to make interfacing brains, skulls, and molds for gel brain models. The skull and brain models can be used to visualize brain tissue inside the skull with the addition of a craniotomy in the custom code, allowing for better preparation for surgeries directly involving the brain. The applications of these methods are designed for surgeries involved in neurological stimulation and recording as well as injection, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

The manipulation and analysis of MRIs as a preoperative craniotomy planning measure has been used successfully in the past2,5,10,16. This process, however, has been greatly enhanced by the addition of the 3D modeling of the brain, skull, and craniotomy. We were able to successfully create an anatomically accurate physical model of the brain that reflected the area of interest for our studies (<...

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A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using ...

This protocol is significant to the neural engineering field because it offers a concise and unimodal procedure designed to enhance the precision and safety of neurosurgery in non-human primates. This technique offers researchers the unique ability to visualize every aspect of their implantation or injection procedure, and ensures that they can enter an operating room as prepared as possible. Visual demonstration of this method is critical because it highlights the impact and clarity that life-size physical models can have on surgical planning.To extract the brain in the magnetic resonance imaging software, open the plugins drop-down menu and select extract brain. Set the extraction intensity threshold at 2.5, 2.7 and the threshold gradient value to zero. After creating a bitmap of the brain image, select build surface under the image menu and input the threshold used to create the bitmap containing the region of interest.Then click okay to create the surface. And save the extracted brain region of interest as a NII or NII. GZ file.To generate a brain model, open the extracted brain file in the appropriate medical image processing software. And in the editor module menu, select threshold effect, adjust the threshold range sliders so the portion of the bitmap containing the brain is highlighted in all three slices. Open the model maker module and in the input volumes dropdown menu, select the bitmap file.Under models, select create new model hierarchy and click apply to create the volume. When the imported mesh brain surface has been imported, select graphic one. And suppress any unnecessary graphic features until only the features containing the brain are remaining in the file.Then, save the files in the PRT format for further manipulation, and as an STL for 3D printing. For brain molding, load the extracted brain model into an appropriate computerated design software program. Under the features section of the insert menu select convert to mesh body and the graphic body of the brain to convert it, and open this sketch tab and click sketch to select the top plane as the sketch plane.Draw a rectangle around the entire hemisphere of interest, and select the extrude boss base feature to extrude a cubic rectangle containing the top part of the brain. Under the features section of the insert menu, select convert to mesh body and select the extruded cube in the solid bodies folder to convert it. To create the negative space, use the combine feature and select the subtract option to subtract the model of the brain from the newly extruded cube.For skull modeling, import the quick MPRAGE MRI into an appropriate matrix manipulation software program as a DICOM file. And use the commands to combine all of the frames into a single 3D matrix as necessary. Ensure that each 2D frame of the matrix displays a coronal slice and use a greater than operator for individual pixel values to threshold the 3D matrix to create a binary mask.Then adjust the threshold such that the skull anatomy is captured by the mask. To remove the musculature layer, iteratively grab a 2D slice from the mask to process each frame from the 3D mask separately, and use the tilde operator to invert the values of the mask. The skull values will be ones.The outside and brain will have values of zero. Add additional empty voxels to the 3D mask until the lowest resolution dimension of the mask is larger by a factor defined by the scale factor. And linearly interpolate values in the mask until the mask fills the new space.Then, export the skull as an STL file, or a similar file type for 3D printing. To create a craniotomy in the 3D skull model, open the MRI file and manually scan back and forth through the 3D matrix to identify the approximate location of the craniotomy using anatomical landmarks found in the macaque brain atlas. To create an agarose mold, pour agarose solution into a full or half hemisphere brain mold and allow the solution to solidify within the mold for about two hours.When the agarose has set, use a spatula to gently remove the gel model from the mold, taking care, not to damage the surface of the mold. For a mock infusion of the agarose gel model, mount a syringe pump onto a stereotaxic arm on a stereotaxic frame and fill a 250 microliter syringe with the ionized water. Load the syringe onto the syringe pump and completely fill the injection cannula with the water.Use the pump driver to load the target volume of food coloring into the syringe for injection. And eject the food coloring until a small bead forms at the tip of the cannula. Dry the bead from the cannula tip and position the gel model under the cannula.Lower the cannula until the tip touches the surface of the gel model, and note the measurements on the stereotaxic arm. Then smoothly and quickly lower the cannula into the gel model to the target injection depth. Making sure that the surface of the gel has sealed around the cannula and run the pump while observing the spread of the die until the target volume has been delivered.Using this protocol, an anatomically accurate physical model of the non-human primate brain can be created. Similarly, an anatomically accurate physical model of the primate skull extracted from magnetic resonance images can also be generated. The physical models of the skull and brain can be combined with a tight interference fit, validating the accuracy of the two models relative to each other and legitimizing the extrapolated MRI analysis data.The insertion of a craniotomy into the skull prior to printing, allows combination of all of the parts of the sample interface for evaluation of the geometry of various components in relation to the skull and brain. For example, in this experiment, the feet of the head post were manipulated and fitted to the curvature of the skull at the location of the implantation prior to the procedure. Resulting in a reduced surgical time from approximately 2.5 hours to one hour from opening to implantation, greatly reducing the risk of operative complications.Agarose mixture brain models can be injected with yellow dye in an area of interest to estimate the volume of the proposed infusion and combining the agarose model with a 3D printed skull can be used to model viral vector injection surgery. Taking into account the variations in skull and brain anatomy in different animals, success in the extraction process will require adjustments in the iterative steps. So this toolbox can be used to reduce risk in non-human primate neurosurgery and the fabrication techniques can enhance the experimental process at the cutting edge of neuroscience.

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4.8K Views.  University of Washington. This protocol is significant to the neural engineering field because it offers a concise and unimodal procedure designed to enhance the precision and safety of neurosurgery in non-human primates. This technique offers researchers the unique ability to visualize every aspect of their implantation or injection procedure, and ensures that they can enter an operating room as prepared as possible. Visual demonstration of this method is critical because it highlights the impact and clarity that lif...