Name: a mri-based toolbox for neurosurgical planning in nonhuman primates
Uploaded: 2020-07-17T14:00:00
Description: Watch this Scientific Journal Video about A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates at JoVE.com

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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Research

JoVE Journal

Bioengineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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 ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

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 ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

In Silico Modeling Method for Computational Aquatic Toxicology of Endocrine Disruptors: A Software-Based Approach Using QSAR Toolbox

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological systems. In particular, quantitative structure-activity relationship (QSAR) models have been increasingly applied to assess the environmental effects of a plethora of toxic materials. In recent years, some more highlighted types of toxicants are endocrine disruptors (EDs, which are chemicals that can interfere with any hormone-related metabolism). Because EDs may significantly affect animal development and reproduction, rapidly predicting the adverse effects of EDs using in silico techniques is required. This study presents an in silico method to generate prediction data on the effects of representative EDs in aquatic vertebrates, particularly fish species. The protocol describes an example utilizing the automated workflow of the QSAR Toolbox software developed by the Organization for Economic Co-operation and Development (OECD) to enable acute ecotoxicity predictions of EDs. As a result, the following are determined: (1) calculation of the numerical correlations between the concentration for 50% of lethality (LC50) and octanol-water partition coefficient (Kow), (2) output performances in which the LC50 values determined in experiments are compared to those generated by computations, and (3) the dependence of estrogen receptor binding affinity on the relationship between Kow and LC50.

Quantitative structure-activity relationship (QSAR) modeling is a representative bioinformatics-assisted method in toxicological screening. This protocol demonstrates how to computationally assess the risks of endocrine disruptors (EDs) in aquatic environments. Utilizing the OECD QSAR Toolbox, the protocol implements an in silico assay for analyzing toxicity of EDs in fish.

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...