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45 ARTICLES PUBLISHED IN JoVE

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Biology

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Kitchener Wilson 1, Jin Yu 1, Andrew Lee 1, Joseph C. Wu 1
1Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

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Biology

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices
Pedro J. Resto 1, Brian Mogen 2, Fan Wu 2, Erwin Berthier 2, David Beebe 2, Justin Williams 2
1Materials Science Program, University of Wisconsin-Madison, 2Department of Biomedical Engineering, University of Wisconsin-Madison

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

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Medicine

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents
Amy N. Turk 1, Stephanie J. Byer 1, Kurt R. Zinn 2, Steven L. Carroll 1,3
1Department of Pathology, University of Alabama at Birmingham - UAB, 2Department of Radiology, University of Alabama at Birmingham - UAB, 3Department of Cell Biology and Neurobiology, University of Alabama at Birmingham - UAB

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

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Neuroscience

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging
Nicholas P. Boyer 1, Chunhe Chen 1, Yiannis Koutalos 1
1Storm Eye Institute, Medical University of South Carolina

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

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Neuroscience

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection
Carlene S. Brandon 1, Christina Voelkel-Johnson 2, Lindsey A. May 3, Lisa L. Cunningham 3
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Microbiology & Immunology, Medical University of South Carolina, 3National Institute on Deafness and Other Communication Disorders, National Institutes of Health

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

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Medicine

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs
Thais Federici 1, Carl V. Hurtig 1, Kentrell L. Burks 1, Jonathan P. Riley 1, Vibhor Krishna 2, Brandon A. Miller 1, Eric A. Sribnick 1, Joseph H. Miller 3, Natalia Grin 1, Jason J. Lamanna 1,4,5, Nicholas M. Boulis 1
1Department of Neurosurgery, Emory University, 2Department of Neuroscience, Medical University of South Carolina, 3Division of Neurosurgery, University of Alabama, Birmingham, 4Department of Biomedical Engineering, Georgia Institute of Technology , 5Department of Biomedical Engineering, Emory University

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

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Neuroscience

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging
Philip O'Herron 1, Zhiming Shen 1, Zhongyang Lu 1, Adrien E. Schramm 1, Manuel Levy 1, Prakash Kara 1
1Department of Neuroscience, Medical University of South Carolina

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

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Medicine

A Contusion Model of Severe Spinal Cord Injury in Rats
Vibhor Krishna 1, Hampton Andrews 1, Xing Jin 2, Jin Yu 1, Abhay Varma 1, Xuejun Wen 3, Mark Kindy 1
1Department of Neuroscience, Division of Neurosurgery, Medical University of South Carolina, 2Bioengineering, Clemson University, 3Clemson-MUSC Bioengineering Joint Program

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

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Medicine

An Orthotopic Bladder Cancer Model for Gene Delivery Studies
Laura Kasman 1, Christina Voelkel-Johnson 1
1Department of Microbiology & Immunology, Medical University of South Carolina

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

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Medicine

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model
Ningfei An 1, Yubin Kang 1
1Division of Hematology-oncology, Department of Medicine, Medical University of South Carolina

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

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Biology

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands
Rushin Shojaii 1, Stephanie Bacopulos 2,3, Wenyi Yang 2,3, Tigran Karavardanyan 4, Demetri Spyropoulos 5, Afshin Raouf 6, Anne Martel 1,4, Arun Seth 2,3
1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

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Engineering

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue
Lucie Sancey 1, Vincent Motto-Ros 2, Shady Kotb 1, Xiaochun Wang 3, François Lux 1, Gérard Panczer 3, Jin Yu 2, Olivier Tillement 1
1ILM-FENNEC UMR 5306, CNRS - Université Lyon 1, 2ILM-PUBLI UMR 5306, CNRS - Université Lyon 1, 3ILM-SOPRANO UMR 5306, CNRS - Université Lyon 1

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 μm and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

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Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys
Liwei Huang 1, An Xiao 1, Andrea Wecker 1, Daniel A. McBride 1, Soo Young Choi 2, Weibin Zhou 3, Joshua H. Lipschutz 2
1Department of Medicine, Eastern Virginia Medical School, 2Department of Medicine, Medical University of South Carolina, 3Department of Pediatrics, University of Michigan

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

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Medicine

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts
Daniel J. Herr 1, Sverre E. Aune 1, Donald R. Menick 1,2
1Department of Medicine/Cardiology, Gazes Cardiac Research Institute, Medical University of South Carolina, 2Ralph H. Johnson VA Medical Center, Medical University of South Carolina

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

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Medicine

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration
Shawn M. Stevens 1, LaShardai N. Brown 2, Paula C. Ezell 3, Hainan Lang 2
1Department of Otolaryngology Head and Neck Surgery, Medical University of South Carolina, 2Department of Pathology and Laboratory Sciences, Medical University of South Carolina, 3Intuitive Surgical, Inc

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

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Bioengineering

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer
Sarah Grace Dennis 1, Thomas Trusk 2, Dylan Richards 3, Jia Jia 3, Yu Tan 3, Ying Mei 3, Stephen Fann 1, Roger Markwald 3, Michael Yost 1
1Department of Surgery, Medical University of South Carolina, 2Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 3Department of Bioengineering, Clemson University

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

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Medicine

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR
Melissa A. Abt 1, Christina L. Grek 2, Gautam S. Ghatnekar 2, Elizabeth S. Yeh 1
1Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 2FirstString Research, Inc.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

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Neuroscience

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo
Tristan Altwegg-Boussac 1,2,3,4, Séverine Mahon 1,2,3,4, Mario Chavez 1,2,3,4, Stéphane Charpier 1,2,3,4, Adrien E. Schramm 1,2,3,4
1Inserm U1127, 2CNRS UMR 7225, 3UPMC Univ Paris 06, UMR S 1127, Sorbonne Universités, 4Institut du Cerveau et de la Moelle épinière (ICM)

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

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Bioengineering

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels
Veronica Rodriguez-Rivera 1, John W. Weidner 2, Michael J. Yost 1
1Department of Surgery - Division of General Surgery, Medical University of South Carolina, 2Department of Chemical Engineering, University of South Carolina

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

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Description of a Novel, Surgically Implanted Neuromodulatory Technique Known As Bilateral Epidural Prefrontal Cortical Stimulation (Epcs) for Treatment-Resistant Depression (TRD)
Nolan R. Williams 4, Jaspreet Pannu *4, Brandon S. Bentzley *4, Thomas Hopkins *1, Bashar W. Badran 1, E. Baron Short 1, Mark S. George 1,2,3, Istvan Takacs 2, Ziad Nahas 5
1Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, 2Department of Neurosciences, Medical University of South Carolina, 3Ralph H. Johnson VA Medical Center, 4Department of Psychiatry and Behavioral Sciences, Stanford University, 5American University of Beirut Medical Center

We describe the implantation of 4 epidural stimulation paddles directly above the dura mater over both the left and right frontopolar and dorsolateral prefrontal cortices. Placement was verified using postoperative computed tomography (CT) coregistered with presurgical magnetic resonance imaging (MRI).

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Developmental Biology

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery
Jui-Tung Liu 1, Mary Paige Lamprecht 1, Stephen A. Duncan 1
1Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

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Bioengineering

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology
Chase A. Arbra *1, Satish N. Nadig *1, Sarah Grace Dennis 1, Sanket Pattanaik 1, Heather A. Bainbridge 1, J. Matthew Rhett 1, Stephen A. Fann 1, Carl Atkinson 1, Michael J. Yost 1
1Department of Surgery, Medical University of South Carolina

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

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Behavior

Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements
Csilla Ari 1, Dominic P. D’Agostino 2,3, David M. Diamond 1,2, Mark Kindy 4,5,6, Collin Park 1, Zsolt Kovács 7
1Department of Psychology, Hyperbaric Neuroscience Research Laboratory, University of South Florida, 2Department of Molecular Pharmacology and Physiology, Metabolic Medicine Research Laboratory, Morsani College of Medicine, University of South Florida, 3Institute for Human and Machine Cognition, 4Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 5James A. Haley VA Medical Center, 6Shriners Hospital for Children, 7Savaria Department of Biology, ELTE Eötvös Loránd University

Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.

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Genetics

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds
Emily Keit 1, Shixuan Chen 1, Hongjun Wang 1, Jingwei Xie 1
1Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

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Neuroscience

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation
Charalambos C. Charalambous 1,2, Jing Nong Liang 3,4, Steve A. Kautz 2,5, Mark S. George 5,6, Mark G. Bowden 2,5,7
1Department of Neurology, New York University School of Medicine, 2Department of Health Sciences and Research, Medical University of South Carolina, 3Department of Physical Therapy, University of Nevada Las Vegas, 4Department of Health Professions, Medical University of South Carolina, 5Ralph H. Johnson VA Medical Center, 6Department of Psychiatry, Medical University of South Carolina, 7Division of Physical Therapy, Medical University of South Carolina

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

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Neuroscience

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations
Bashar W. Badran *1,2,3, Alfred B. Yu *2, Devin Adair 1, Georgia Mappin 3, William H. DeVries 3, Dorothea D. Jenkins 4, Mark S. George 3,5,6, Marom Bikson 1
1Department of Biomedical Engineering, City College of New York, 2U.S. Army Research Laboratory, Aberdeen Proving Ground, 3Brain Stimulation Laboratory, Department of Psychiatry, Medical University of South Carolina, 4Department of Pediatrics, Medical University of South Carolina, 5Department of Neurology, Medical University of South Carolina, 6Ralph H. Johnson VA Medical Center

A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

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Biology

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents
John Malysz 1, Eric S. Rovner 2, Robert Wake 3, Georgi V. Petkov 1,3,4
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 2Department of Urology, Medical University of South Carolina, 3Department of Urology, College of Medicine, University of Tennessee Health Science Center, 4Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

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Bioengineering

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye
Marwa Belhaj 1, Balasubramaniam Annamalai 2, Nathaniel Parsons 2, Andrew Shuler 1, Jay Potts 1, Bärbel Rohrer 2,3,4
1Department of Cell Biology, University of South Carolina, 2Department of Ophthalmology, Division of Research, Medical University of South Carolina, 3Department of Neuroscience, Division of Research, Medical University of South Carolina, 4Division of Research, Ralph H. Johnson VA Medical Center

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

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Neuroscience

Cochlear Surface Preparation in the Adult Mouse
Qiao-Jun Fang 1,2, Fan Wu 1, Renjie Chai 2, Su-Hua Sha 1
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2MOE Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

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Cancer Research

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad
Lauren B. Shunkwiler 1, Jill D. Haag 2, Michael N. Gould 2, Bart M. G. Smits 1
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

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Neuroscience

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve
Nicholas P. Boyer 1, Maite Azcorra 1,2, Peter Jung 3,4, Anthony Brown 1
1Department of Neuroscience, The Ohio State University, 2Present address: Interdepartmental Neuroscience Graduate Program and Department of Neurobiology, Northwestern University, 3Quantitative Biology Institute, Ohio University, 4Department of Physics and Astronomy, Ohio University

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

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Medicine

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion
Wenyu Gou *1, Lindsay Swaby *1, A. Marissa Wolfe 2, William P. Lancaster 1,2, Katherine A. Morgan 1, Hongjun Wang 1,2
1Department of Surgery, Medical University of South Carolina, 2Ralph H. Johnson Veterans Affairs Medical Center

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

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Medicine

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice
Wenyu Gou *1, Wanxing Cui *2, Yuki Cui 2, Hongjun Wang 1,3
1Department of Surgery, Medical University of South Carolina, 2Georgetown University, 3Ralph H. Johnson Veterans Affairs Medical Center

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

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JoVE Core

Standing Neurophysiological Assessment of Lower Extremity Muscles Post-Stroke
John Harvey Kindred 1,2, Christian Finetto 3, Jasmine Jamilah Cash 3, Mark Goodman Bowden 1,2,3
1Department of Research and Development, Ralph H. Johnson Veterans Administration Medical Center, 2College of Health Professions, Department of Rehabilitation Sciences, Division of Physical Therapy, Medical University of South Carolina, 3College of Health Professions, Department of Health Sciences and Research, Medical University of South Carolina

This protocol describes the process for performing a neurophysiological assessment of the lower extremity muscles, tibialis anterior and soleus, in a standing position using TMS in people post-stroke. This position provides a greater probability of eliciting a post-stroke TMS response and allows for the use of reduced stimulator power during neurophysiological assessments.

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Developmental Biology

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids
Na Qu 1, Abdelkader Daoud 1, Braxton Jeffcoat 1, Jorge O. Múnera 1
1Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

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Cancer Research

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells
Jody Fromm Longo 1, Stephanie N. Brosius 2,3, Steven L. Carroll 1
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Pediatrics and Neurology, Perelman School of Medicine of the University of Pennsylvania, 3Division of Child Neurology, Children's Hospital of Philadelphia

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

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Medicine

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents
Morgan A. Hill 1, Jennie H. Kwon 1, Brielle Gerry 1, Minoo Kavarana 1, Satish N. Nadig *1, Taufiek K. Rajab *1
1Department of Surgery, Medical University of South Carolina

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

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Biology

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion
Chao E *1, Liqiang Dai *1,2, Jiaqi Tian 3,4, Lin-Tai Da 4, Jin Yu 5,6,7
1Beijing Computational Science Research Center, 2Shenzhen JL Computational Science and Applied Research Institute, 3School of Medical Informatics and Engineering, Xuzhou Medical University, 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 5Department of Physics and Astronomy, University of California, Irvine, 6Department of Chemistry, University of California, Irvine, 7NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine

The goal of this protocol is to reveal structural dynamics of one-dimensional diffusion of protein along DNA, using a plant transcription factor WRKY domain protein as an exemplary system. To do this, both atomistic and coarse-grained molecular dynamics simulations along with extensive computational samplings have been implemented.

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Education

The Evoked Potential Operant Conditioning System (EPOCS): A Research Tool and an Emerging Therapy for Chronic Neuromuscular Disorders
N. Jeremy Hill 1,2, Disha Gupta 1,2, Amir Eftekhar 1, Jodi A. Brangaccio 1, James J. S. Norton 1,2, Michelle McLeod 3, Tim Fake 1, Jonathan R. Wolpaw 1,2,4, Aiko K. Thompson 3
1National Center for Adaptive Neurotechnologies, Stratton VA Medical Center, 2Electrical & Computer Engineering Department, State University of New York at Albany, 3College of Health Professions, Medical University of South Carolina, 4Department of Biomedical Sciences, State University of New York at Albany

The Evoked Potential Operant Conditioning System aids scientific investigation of sensorimotor function and can administer targeted neurobehavioral training that can impact sensorimotor rehabilitation in neuromuscular disorders. This article describes its capabilities and illustrates its application in modifying a simple spinal reflex to achieve lasting improvement in motor function.

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Cancer Research

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor
Brittany Turner-Ivey 1, Jody Fromm Longo 2, Dorea P. Jenkins 2, Stephen T. Guest 3, Steven L. Carroll 1,2
1Hollings Cancer Center, Medical University of South Carolina, 2Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 3Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

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Cancer Research

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models
Dorea P. Jenkins 1, Brittany Turner-Ivey 1,2, Jody Fromm Longo 1, Steven L. Carroll 1,2
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Hollings Cancer Center, Medical University of South Carolina

We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGFβ3 mouse model.

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Medicine

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection
Jen X. Xu 1, Frank Z. Xu 2, Amelia Furbish 1, Yuri K. Peterson 1, Patrick M. Woster 1, Alicia M. Braxton 3
1Drug Discovery and Biomedical Sciences, Medical University of South Carolina, 2Department of Biomedical Science, The University of Alabama at Birmingham, 3Department of Comparative Medicine, Medical University of South Carolina

Dunkin-Hartley guinea pigs are an established animal model for osteoarthritis research. Such studies may benefit from intra-articular injections for various reasons, including investigating novel agents or treating disease. We describe a methodology for intra-articular knee injections in Guinea pigs and subsequent micro-computed tomography analysis assessing arthritis-associated knee changes.

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Neuroscience

PyDesigner v1.0: A Pythonic Implementation of the DESIGNER Pipeline for Diffusion Magnetic Resonance Imaging
Siddhartha Dhiman *1, Reyna E. Hickey *2, Kathryn E. Thorn *2, Hunter G. Moss 1,3, Emilie T. McKinnon 1,3, Vitria Adisetiyo 1, Benjamin Ades-Aron 4, Jens H. Jensen 1,3,5, Andreana Benitez 2,3
1Department of Neuroscience, Medical University of South Carolina, 2Department of Neurology, Medical University of South Carolina, 3Center for Biomedical Imaging, Medical University of South Carolina, 4Center for Biomedical Imaging, Department of Radiology, New York University School of Medicine, 5Department of Radiology and Radiological Science, Medical University of South Carolina

Here we present PyDesigner, a Python-based diffusion magnetic resonance imaging (dMRI) processing pipeline capable of correcting for typical dMRI artifacts and producing diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), fiber ball imaging (FBI), microstructure modeling (white matter integrity [WMTI] and fiber ball white matter [FBWM]), and tractography outputs.

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Biochemistry

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells
Russell Spencer-Smith 1,2,3
1Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 2Hollings Cancer Center, Medical University of South Carolina, 3Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

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Biology

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue
Sarah A. Dooley *1, Rachel Stubler *1, Rachel M. Edens 1, Piper R. McKee 1, Jordan N. Rucker 1, Amy Engevik 1
1Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina

The intestine is vital for digestion and absorption. Each region-duodenum, jejunum, ileum, colon-serves distinct functions due to unique cellular structures. Studying intestinal physiology demands meticulous tissue analysis. This protocol outlines tissue fixation and processing using the Swiss roll technique, ensuring accurate immunostaining through proper tissue preservation and orientation.

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