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.

<ol>
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The authors have no conflicts of interest to disclose at this time.

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 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 (<...

Watch this Scientific Journal Video about A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates at JoVE.com

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 ...

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Research

JoVE Journal

Bioengineering

4.9K Views.  University of Washington. 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.

Video: A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

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 ...

Cancer Research

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

Biology

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Medicine

Automated Segmentation of Cortical Grey Matter from T1-Weighted MRI Images

Within neuroimaging research, a number of recent studies have discussed the impact of between-study differences in volumetric findings that are thought to result from the use of different segmentation tools to generate brain volumes. Here, processing pipelines for seven automated tools that can be used to segment grey matter within the brain are presented. The protocol provides an initial step for researchers aiming to find the most accurate method for generating grey matter volumes from T1-weighted MRI scans. Steps to undertake detailed visual quality control are also included in the manuscript. This protocol covers a range of potential segmentation tools and encourages users to compare the performance of these tools within a subset of their data before selecting one to apply to a full cohort. Furthermore, the protocol may be further generalized to the segmentation of other brain regions.

This protocol describes the process of applying seven different automated segmentation tools to structural T1-weighted MRI scans to delineate grey matter regions that can be used for the quantification of grey matter volume.

Within neuroimaging research, a number of recent studies have discussed the impact of between-study differences in volumetric findings that are thought to ...

Neuroscience

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, neuroanesthesia, and neurosurgery. Patients who have tumors or epileptogenic tissue in eloquent brain areas are studied preoperatively with functional and structural MRI, and intraoperatively with direct electrical stimulation mapping. Post-operative neural and cognitive outcome measures fuel basic science studies about the factors that mediate good versus poor outcome after surgery, and how brain mapping can be further optimized to ensure the best outcome for future patients. In this article, we describe the interdisciplinary workflow that allows our team to meet the synergistic goals of optimizing patient outcome and advancing scientific understanding of the human brain.

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the ...

Automated 3D Brain and Skull Modeling from MRI for NHP Neurosurgical Planning

A Neural Implant Design Toolbox for Nonhuman Primates

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.

Author Spotlight: Streamlined Brain and Skull Modeling for Enhanced Neurosurgical Planning in NHP Research

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.

This paper describes an in-house method of 3D brain and skull modeling from magnetic resonance imaging (MRI) tailored for nonhuman primate (NHP) ...

Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of benign and malignant lesions through a natural anatomical extra-cranial pathway, represented by the nasal cavities, avoiding brain retraction and neurovascular manipulation. This is reflected by the patients' prompt clinical recovery and the low risk of permanent neurological sequelae, representing the main caveat of conventional skull base surgery. This surgery must be tailored to each specific case, considering its features and relationship with surrounding neural structures, mostly based on preoperative neuroimaging. Advanced MRI techniques, such as tractography, have been rarely adopted in skull base surgery due to technical issues: lengthy and complicated processes to generate reliable reconstructions for inclusion in the neuronavigation system. 
This paper aims to present the protocol implemented in the institution and highlights the synergistic collaboration and teamwork between neurosurgeons and the neuroimaging team (neurologists, neuroradiologists, neuropsychologists, physicists, and bioengineers) with the final goal of selecting the optimal treatment for each patient, improving the surgical results and pursuing the advancement of personalized medicine in this field.

We present a protocol to integrate diffusion MRI tractography in patient work-up to endoscopic endonasal surgery for a skull base tumor. The methods for adopting these neuroimaging studies in the pre- and intra-operative phases are described.

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of ...

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 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 ...

High-Precision TUS: Planning and Execution with Acoustic and Thermal Simulations

Pipeline for Planning and Execution of Transcranial Ultrasound Neuromodulation Experiments in Humans

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique capable of manipulating both cortical and subcortical structures with high precision. Conducting experiments involving humans necessitates careful planning of acoustic and thermal simulations. This planning is essential to adjust for bone interference with the ultrasound beam's shape and trajectory and to ensure TUS parameters meet safety requirements. T1- and T2-weighted, along with zero-time echo (ZTE) magnetic resonance imaging (MRI) scans with 1 mm isotropic resolution, are acquired (alternatively computed tomography x-ray (CT) scans) for skull reconstruction and simulations. Target and trajectory mapping are performed using a neuronavigational platform. SimNIBS is used for the initial segmentation of the skull, skin, and brain tissues. Simulation of TUS is carried over with the BabelBrain tool, which uses the ZTE scan to produce synthetic CT images of the skull to be converted into acoustic properties. We use a phased array ultrasound transducer with electrical steering capabilities. Z-steering is adjusted to ensure that the target depth is reached. Other transducer configurations are also supported in the planning tool. Thermal simulations are run to ensure temperature and mechanical index requirements are within the acoustic guidelines for TUS in human subjects as recommended by the FDA. During TUS delivery sessions, a mechanical arm assists in the movement of the transducer to the required location using a frameless stereotactic localization system.

Author Spotlight: Advancing Human Brain Modulation &#8211; Optimized Protocols for Transcranial Ultrasound Stimulation Experiments

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique that requires careful planning of acoustic and thermal simulations. The methodology describes an image processing and ultrasound simulation pipeline for efficient, user-friendly, streamlined planning for human TUS experimentation.

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique capable of manipulating both cortical and subcortical ...