We would like to thank Toni Haun, Keith Vogel and Shawn Fisher for their technical help and support. This work was supported by the University of Washington Mary Gates Endowment (R.I.), National Institute of Health NIH 5R01NS116464 (T.B., A.Y.), NIH R01 NS119395 (D.J.G., A.Y), the Washington National Primate Research Center (WaNPRC, NIH P51 OD010425, U42 OD011123), the Center for Neurotechnology (EEC-1028725, Z.A., D.J.G.) and Weill Neurohub (Z. I.).

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

<ol>
	<li>Mitchell, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continued+need+for+nonhuman+primate+neuroscience+research.">Continued need for nonhuman primate neuroscience research.</a> Current Biology. 28 (20), R1186-R1187 (2018).</li><li>Stanis, N., Khateeb, K., Zhou, J., Wang, R. K., Yazdan-Shahmorad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+study+ischemic+stroke+by+photothrombotic+lesioning+in+the+cortex+of+nonhuman+primates.">Protocol to study ischemic stroke by photothrombotic lesioning in the cortex of nonhuman primates.</a> STAR Protocols. 4 (3), 102496 (2023).</li><li>Tremblay, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+resource+for+nonhuman+primate+optogenetics.">An open resource for nonhuman primate optogenetics.</a> Neuron. 108 (6), 1075-1090 (2020).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+electrical+stimulation+following+ischemic+stroke+in+nonhuman+primates.">Neuroprotective effects of electrical stimulation following ischemic stroke in nonhuman primates.</a> 2022 44th Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). 2022, 3085-3088 (2022).</li><li>Yao, Z., Yazdan-Shahmorad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+model+for+estimating+the+scale+of+photochemically+induced+ischemic+stroke.">A quantitative model for estimating the scale of photochemically induced ischemic stroke.</a> 2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC). 2018, 2744-2747 (2018).</li><li>Yazdan-Shahmorad, A., Silversmith, D. B., Kharazia, V., Sabes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+cortical+reorganization+using+optogenetics+in+nonhuman+primates.">Targeted cortical reorganization using optogenetics in nonhuman primates.</a> eLife. 7, e31034 (2018).</li><li>Macknik, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+circuit+and+cellular+imaging+methods+in+nonhuman+primates.">Advanced circuit and cellular imaging methods in nonhuman primates.</a> The Journal of Neuroscience. 39 (42), 8267-8274 (2019).</li><li>Griggs, D. J., Belloir, T., Yazdan-Shahmorad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+neural+interfaces+for+optogenetic+actuators+and+sensors+in+non-human+primates.">Large-scale neural interfaces for optogenetic actuators and sensors in non-human primates.</a> SPIE BiOS. , (2021).</li><li>Yazdan-Shahmorad, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Large-scale+interface+for+optogenetic+stimulation+and+recording+in+nonhuman+primates.">A Large-scale interface for optogenetic stimulation and recording in nonhuman primates.</a> Neuron. 89 (5), 927-939 (2016).</li><li>Ruiz, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+through+windows+on+the+brain+in+the+nonhuman+primate.">Optogenetics through windows on the brain in the nonhuman primate.</a> Journal of Neurophysiology. 110 (6), 1455-1467 (2013).</li><li>Griggs, D. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+non-invasive+neuroimaging+to+enhance+the+care,+well-being+and+experimental+outcomes+of+laboratory+nonhuman+primates+(monkeys).">Using non-invasive neuroimaging to enhance the care, well-being and experimental outcomes of laboratory nonhuman primates (monkeys).</a> NeuroImage. 228, 117667 (2021).</li><li>Ahmed, Z., Agha, N., Trunk, A., Berger, M., Gail, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+guide+for+skull+extraction+and+custom-fitting+of+implants+to+continuous+and+discontinuous+skulls.">Universal guide for skull extraction and custom-fitting of implants to continuous and discontinuous skulls.</a> eNeuro. 9 (3), (2022).</li><li>Blonde, J. 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MathWorks Available from: <a target="_blank" href="https://www.mathworks.com/matlabcentral/fileexchange/68794-make-stl-of-3d-array-optimal-for-3d-printing">https://www.mathworks.com/matlabcentral/fileexchange/68794-make-stl-of-3d-array-optimal-for-3d-printing</a> (2018)</li><li>. stlwrite - Write binary or ascii STL file Available from: <a target="_blank" href="https://www.mathworks.com/matlabcentral/fileexchange/36770-stlwrite-write-binary-or-ascii-stl-file">https://www.mathworks.com/matlabcentral/fileexchange/36770-stlwrite-write-binary-or-ascii-stl-file</a> (2023)</li><li>Michikawa, T., Suzuki, H., Moriguchi, M., Ogihara, N., Kondo, O., Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+extraction+of+endocranial+surfaces+from+CT+images+of+crania.">Automatic extraction of endocranial surfaces from CT images of crania.</a> PLoS One. 12 (4), 0168516 (2017).</li><li>Overton, J. A., Cooke, D. F., Goldring, A. B., Lucero, S. A., Weatherford, C., Recanzone, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+acrylic-free+implants+in+nonhuman+primates+for+neuroscience+research.">Improved methods for acrylic-free implants in nonhuman primates for neuroscience research.</a> Journal of Neurophysiology. 118 (6), 3252-3270 (2017).</li><li>Ortiz-Rios, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+MRI-compatible+implants+in+nonhuman+primates.">Improved methods for MRI-compatible implants in nonhuman primates.</a> Journal of Neuroscience Methods. 308, 377-389 (2018).</li></ol>

Nothing to disclose.

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 research13,15,25. In comparison to other current methods of NHP implant planning and design25,29,30<...

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 dysfunction1,2,3,4,5,6,7,8. 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 windows9,10,11,12,13,14,15,16,17,18,19,20,21,22,23. In NHPs, cranial windows are often achieved with a chamber and an artificial dura to protect the brain and support long-term experimentation8,10,12,17,18,24,25,26,27. Likewise, headposts often accompany chambers to stabilize and align the head during experiments14,15,25,26,28,29,30. 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 surgery25,29 and estimating the skull curvature by fitting circles to coronal and sagittal slices of magnetic resonance (MR) scans9,12 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 longevity9,29,30,32. 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) machines30,32,33 and&#160;even computer numerical control (CNC) milling machines for developing implant prototypes25,29,32,34. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Printing Software (Simplify 3D) (Paid)</td>
			<td>Simplify3D</td>
			<td>Version 4.1</td>
			<td>Used for 3D printing using MakerGear printer</td>
		</tr>
		<tr>
			<td>C-Clamp</td>
			<td>Bessey</td>
			<td>CM22</td>
			<td>Used for artificial dura fabrication, 2-1/2 Inch Capacity, 1-3/8 Inch Throat</td>
		</tr>
		<tr>
			<td>Formlabs Form 3+ 3D Printer</td>
			<td>Formlabs</td>
			<td>Form 3+</td>
			<td>Used for precise 3D printing</td>
		</tr>
		<tr>
			<td>MakerGear M2 3D Printer</td>
			<td>MakerGear</td>
			<td>M2 revG</td>
			<td>Used for 3D printing implant prototypes</td>
		</tr>
		<tr>
			<td>MATLAB (Paid)</td>
			<td>MathWorks</td>
			<td>R2021b</td>
			<td>Used for brain and skull isolation, virtual craniotomy visualization and skull STL reduction</td>
		</tr>
		<tr>
			<td>Phillips Acheiva MRI System</td>
			<td>Philips</td>
			<td>4522 991 19391</td>
			<td>Used for non-human primate imaging</td>
		</tr>
		<tr>
			<td>Photopolymer Resin</td>
			<td>Formlabs</td>
			<td>FLGPGR04</td>
			<td>1L, Grey, used for precise 3D prints with Formlabs printer&#160;</td>
		</tr>
		<tr>
			<td>PreForm Print Preparation Software</td>
			<td>Formlabs</td>
			<td>Version 2.17.0</td>
			<td>Used for 3D printing with Formlabs printer&#160;</td>
		</tr>
		<tr>
			<td>Printing Filament (PLA)</td>
			<td>MatterHackers</td>
			<td>88331</td>
			<td>PLA 1.75 mm White. Used for 3D printing with MakerGear printer</td>
		</tr>
		<tr>
			<td>Silicone CAT-1300</td>
			<td>Shin-Etsu</td>
			<td></td>
			<td>Used for artificial dura fabrication</td>
		</tr>
		<tr>
			<td>Silicone KE1300-T</td>
			<td>Shin-Etsu</td>
			<td></td>
			<td>Used for artificial dura fabrication</td>
		</tr>
		<tr>
			<td>SolidWorks (Paid)</td>
			<td>Dassault Systems</td>
			<td>2020</td>
			<td>Used for chamber and headpost design</td>
		</tr>
		<tr>
			<td>Syn.Flex-S Multicoil</td>
			<td>Philips</td>
			<td>45221318123</td>
			<td>Used for non-human primate imaging</td>
		</tr>
	</tbody>
</table>

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.

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). Additi...

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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.

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) ...

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Research

JoVE Journal

Bioengineering

1.0K Views.  University of Washington. 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.

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

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...