West Virginia University View Institution's Website 32 articles published in JoVE Bioengineering Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles Olivia L. Rose1, Myra Arnold2,3, Cerasela-Zoica Dinu1 1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Anthropology for Energy, West Virginia University, 3Department of Hospitality and Tourism, West Virginia University The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique. Chemistry Synthesis of a Borylated Ibuprofen Derivative Through Suzuki Cross-Coupling and Alkene Boracarboxylation Reactions Steven W. Knowlden*1, Randika T. Abeysinghe*1, Amanda D. Swistok1, Alexis C. Ravenscroft1, Brian V. Popp1 1C. Eugene Bennett Department of Chemistry, West Virginia University The present protocol describes a detailed benchtop catalytic method that yields a unique borylated derivative of ibuprofen. Biology Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence Alyson Nguyen*1, Keith Battle*2,3, Sunita S. Paudel*2,3, Ningyong Xu2, Jessica Bell3, Linn Ayers3, Cassandra Chapman4, Ajay P. Singh5, Srinivas Palanki6, Thomas Rich3,7, Diego F. Alvarez8, Troy Stevens2,3, Dhananjay T. Tambe3,4,7 1Biomedical Sciences, Pat Capps Covey College of Allied Health Professions, University of South Alabama, 2Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, 3Center for Lung Biology, College of Medicine, University of South Alabama, 4William B. Burnsed Jr. Mechanical, Aerospace, and Biomedical Engineering Department, College of Engineering, University of South Alabama, 5Mitchell Cancer Institute, College of Medicine, University of South Alabama, 6Department of Chemical and Biomedical Engineering, West Virginia University, 7Department of Pharmacology, College of Medicine, University of South Alabama, 8Department of Physiology & Pharmacology, College of Osteopathic Medicine, Sam Houston State University The Integrative Toolkit to Analyze Cellular Signals (iTACS) platform automates the process of simultaneously measuring a wide variety of chemical and mechanical signals in adherent cells. iTACS is designed to facilitate community-driven development and enable researchers to use all platform features regardless of their educational background. Engineering Development and Functionalization of Electrolyte-Gated Graphene Field-Effect Transistor for Biomarker Detection Sakib Ishraq1, Jianbo Sun2, Yuxin Liu1 1Lane Department of Computer Science and Electrical Engineering, West Virginia University, 2Beijing Graphene Institute The present protocol demonstrates the development of electrolyte-gated graphene field-effect transistor (EGGFET) biosensor and its application in biomarker immunoglobulin G (IgG) detection. Biochemistry Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes Schuyler D. Vickers*1, Dominique C. Saporito*1, Roberta Leonardi1 1Department of Biochemistry, School of Medicine, West Virginia University Fatty acid β-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid. Cancer Research Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique Pavan Rao1, Carl R. Schmidt1, Brian A. Boone1,2 1Division of Surgical Oncology, Department of Surgery, West Virginia University, 2Department of Microbiology, Immunology and Cell Biology, West Virginia University We present our operative approach to robot assisted distal pancreatectomy, splenectomy, and celiac axis resection (DP-CAR), demonstrating that the procedure is safe and feasible with proper planning, patient selection, and surgeon experience. Biology Irradiator Commissioning and Dosimetry for Assessment of LQ α and β Parameters, Radiation Dosing Schema, and in vivo Dose Deposition Samuel A. Sprowls*1, Vincenzo J. Pizzuti*1,3, William Pentz1, Divine C. Nwafor3,4, R. Alfredo C. Siochi2,3, Paul R. Lockman1 1Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, 2Radiation Oncology, West Virginia University, 3School of Medicine, West Virginia University, 4Department of Neuroscience, West Virginia University Radiation dosimetry provides a technique for enhancing the accuracy of preclinical experiments and ensuring that the radiation doses delivered are closely related to clinical parameters. This protocol describes steps to be taken at each phase during preclinical radiation experiments to ensure proper experimental design. Neuroscience Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents Aric F. Logsdon*1,2, Brandon P. Lucke-Wold*3, Ryan C. Turner*4, Sean M. Collins5, Evan L. Reeder5, Jason D. Huber4, Charles L. Rosen6, Matthew J. Robson*5, Florian Plattner*7 1Geriatrics Research Education and Clinical Center, Veterans Affairs, 2Division of Gerontology and Geriatric Medicine, University of Washington, 3Department of Neurosurgery, University of Florida, 4Department of Neurosurgery, West Virginia University, 5Division of Pharmaceutical Sciences, University of Cincinnati, 6Central Illinois Neuro Health Sciences, Bloomington, IL, 7Neuro-research, Dallas, TX We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury. Immunology and Infection Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment Kelly L. Monaghan*1, Breanne Y. Farris*1, Wen Zheng1, Edwin C. K. Wan1,2,3 1Department of Microbiology, Immunology, and Cell Biology, West Virginia University, 2Department of Neuroscience, West Virginia University, 3Rockefeller Neuroscience Institute, West Virginia University This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke. Bioengineering Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate Celia Martinez de la Torre1, Margaret F. Bennewitz1 1Department of Chemical and Biomedical Engineering, West Virginia University This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment. Medicine Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction Anna L. Lang1,2, William T. Goldsmith3,4, Regina D. Schnegelberger5,6, Gavin E. Arteel6,7, Juliane I. Beier6,7 1Department of Pharmacology and Toxicology, University of Louisville, 2Hepatobiology and Toxicology Program, University of Louisville, 3Department of Physiology and Pharmacology, West Virginia University, 4Center for Inhalation Toxicology, West Virginia University, 5Department of Pharmacology and Chemical Biology, University of Pittsburgh, 6Pittsburgh Liver Research Center, University of Pittsburgh, 7Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe. Behavior Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients Shuo Wang1, Nand Chandravadia2, Adam N. Mamelak2, Ueli Rutishauser2,3,4 1Department of Chemical and Biomedical Engineering, and Rockefeller Neuroscience Institute, West Virginia University, 2Departments of Neurosurgery and Neurology, Cedars-Sinai Medical Center, 3Center for Neural Science and Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Division of Biology and Biological Engineering, California Institute of Technology We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search. Environment Necropsy-based Wild Fish Health Assessment Vicki S. Blazer1, Heather L. Walsh2, Ryan P. Braham1, Cheyenne Smith2 1National Fish Health Research Laboratory, Leetown Science Center, U.S. Geological Survey, 2School of Natural Resources, West Virginia University The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses. Cancer Research In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment Andrey A. Bobko1,2, Timothy D. Eubank1,3, Benoit Driesschaert1,2, Valery V. Khramtsov1,2 1In Vivo Multifunctional Magnetic Resonance center, Robert C. Byrd Health Sciences Center, West Virginia University, 2Department of Biochemistry, West Virginia University School of Medicine, 3Department of Microbiology, Immunology & Cell Biology, West Virginia University School of Medicine Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer. Engineering Resonance Fluorescence of an InGaAs Quantum Dot in a Planar Cavity Using Orthogonal Excitation and Detection Disheng Chen1, Gary R. Lander1, Edward B. Flagg1 1Department of Physics and Astronomy, West Virginia University Resonant excitation of a single self-assembled quantum dot can be achieved using an excitation mode orthogonal to the fluorescence collection mode. We demonstrate a method using the waveguide and Fabry-Perot modes of a planar microcavity surrounding the quantum dots. The method allows complete freedom in the detection polarization. Neuroscience Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models Holly C. Hunsberger1, Sharay E. Setti2, Ryan T. Heslin2, Jorge E. Quintero3, Greg A. Gerhardt3, Miranda N. Reed2 1Department of Psychology, West Virginia University, 2Department of Drug Discovery & Development, Auburn University, 3Department of Neuroscience, University of Kentucky Medical Center Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA). Bioengineering Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior Kai Wang1, Kam W. Leong2, Yong Yang1 1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented. Neuroscience Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells Stephanie L. Rellick1,2,4, Heng Hu1,3,4, James W. Simpkins1,4, Xuefang Ren1,3,4 1Department of Physiology and Pharmacology, West Virginia University, 2Mitochondrial Evaluation Core, West Virginia University, 3Experimental Stroke Core, West Virginia University, 4Center for Basic and Translational Stroke Research, West Virginia University Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells. Bioengineering Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo Joshua R. Biller1,2, Deborah G. Mitchell1, Mark Tseytlin3,4, Hanan Elajaili1, George A. Rinard5, Richard W. Quine6, Sandra S. Eaton1, Gareth R. Eaton1 1Department of Chemistry and Biochemistry, University of Denver, 2Magnetic Imaging Group, Applied Physics Division, Physical Measurements Laboratory, National Institute of Standards and Technology, 3Department of Radiology, Geisel School of Medicine, Dartmouth University, 4Department of Biochemistry, West Virginia University, 5Department of Electrical and Computer Engineering, University of Denver, 6Department of Engineering, University of Denver A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency. Environment Watershed Planning within a Quantitative Scenario Analysis Framework Eric R. Merriam1, J. Todd Petty1, Michael P. Strager2 1Division of Forestry and Natural Resources, West Virginia University, 2Division of Resource Management, West Virginia University There is a critical need for tools and methodologies capable of managing aquatic systems in the face of uncertain future conditions. We provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework. Engineering Design and Use of a Full Flow Sampling System (FFS) for the Quantification of Methane Emissions Derek R. Johnson1,2, April N. Covington1,2, Nigel N. Clark1 1Mechanical and Aerospace Engineering Department, West Virginia University, 2Center for Alternative Fuels, Engines, and Emissions, West Virginia University We have designed, developed, and implemented a novel full flow sampling system (FFS) for quantification of methane emissions and greenhouse gases from across the natural gas supply chain. Biology Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production Sulei Xu*1, Xiang Li*1, Yuxin Liu2, Pingnian He1 1Department of Cellular and Molecular Physiology, College of Medicine, Penn State University, 2Lane Department of Computer Science and Electrical Engineering, West Virginia University Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels. Behavior An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents Jefferson C. Frisbee1, Steven D. Brooks1, Shyla C. Stanley1, Alexandre C. d'Audiffret2 1Department of Physiology and Pharmacology, West Virginia University Health Sciences Center, 2Division of Vascular and Endovascular Surgery, Center for Cardiovascular and Respiratory Sciences, West Virginia University Health Sciences Center The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender. Behavior Multifunctional Setup for Studying Human Motor Control Using Transcranial Magnetic Stimulation, Electromyography, Motion Capture, and Virtual Reality William J. Talkington1, Bradley S. Pollard1, Erienne V. Olesh1, Valeriya Gritsenko1 1Department of Human Performance and Applied Exercise Science, Division of Physical Therapy, West Virginia University Transcranial magnetic stimulation, electromyography, and 3D motion capture are commonly used non-invasive techniques for investigating neuromuscular function in humans. In this paper, we describe a protocol that synchronously samples data generated by all three of these tools along with the unique addition of virtual reality stimulus presentation and feedback. Medicine High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes Elyse L. Walk*1,3, Sarah L. McLaughlin*2,3, Scott A. Weed1,3 1Department of Neurobiology and Anatomy, West Virginia University, 2Animal Models and Imaging Facility, West Virginia University, 3Mary Babb Randolph Cancer Center, West Virginia University This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated. Behavior Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment Daniel S. Weitzner1, Elizabeth B. Engler-Chiurazzi2, Linda A. Kotilinek3, Karen Hsiao Ashe3,4,5, Miranda Nicole Reed1,6 1Department of Psychology, Behavioral Neuroscience, West Virginia University, 2Department of Physiology and Pharmacology, West Virginia University, 3Department of Neurology, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, 4Department of Neuroscience, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, 5GRECC, VA Medical Center, 6Center for Neuroscience, Center for Basic and Translational Stroke Research, West Virginia University This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences. Bioengineering Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip Xiang Li1, Samantha Marie Mearns1, Manuela Martins-Green2, Yuxin Liu1 1Lane Department of Computer Science and Electrical Engineering, West Virginia University, 2Department of Cell Biology and Neuroscience, University of California at Riverside A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging and can be applied for microvascular research. Biology Whole-Body Nanoparticle Aerosol Inhalation Exposures Jinghai Yi1,2, Bean T. Chen3, Diane Schwegler-Berry3, Dave Frazer3, Vince Castranova3, Carroll McBride1, Travis L. Knuckles1,2, Phoebe A. Stapleton1,2, Valerie C. Minarchick1,2, Timothy R. Nurkiewicz1,2 1Department of Physiology and Pharmacology, School of Medicine, West Virginia University, 2Center for Cardiovascular and Respiratory Sciences, West Virginia University, 3National Institute for Occupational Safety and Health A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation. Immunology and Infection PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP Hongshuai Li1,2, Bingyun Li1,3,4 1Department of Orthopaedics, School of Medicine, West Virginia University, 2Department of Orthopaedics, Stem Cell Research Center, University of Pittsburgh, 3WVNano Initiative, 4Mary Babb Randolph Cancer Center Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro. Biology Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts Karen H. Martin1, Karen E. Hayes1, Elyse L. Walk1, Amanda Gatesman Ammer1, Steven M. Markwell1, Scott A. Weed1 1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields. Biology The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity Joshua T. Butcher1, Adam G. Goodwill1, Jefferson C. Frisbee1 1Department of Physiology and Pharmacology, Center for Cardiovascular and Respiratory Sciences, West Virginia University An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics. Medicine Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma Amanda Gatesman Ammer1, Karen E. Hayes1, Karen H. Martin1, Lingqing Zhang2, George A. Spirou3, Scott A. Weed1 1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, 2Sensory Neuroscience Research Center, West Virginia University, 3Departments of Otolaryngology and Physiology, Center for Neuroscience, West Virginia University A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.