University of Cincinnati View Institution's Website 27 articles published in JoVE Biology Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens Renata Belisário1, Maria F. Torres1,2, Ester A. S. Buiate1,3, Katia V. Xavier1,4, Etta M. Nuckles1, Lisa J. Vaillancourt1 1Department of Plant Pathology, University of Kentucky, 2Department of Biological Sciences, University of Cincinnati, 3Bayer Crop Science, 4Everglades Research and Education Center, University of Florida This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues. Bioengineering Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip Varone Antonio1, Adya Panchal2, Magdalena Kasendra3, Barrile Riccardo2,3 1Merk Sharp & Dohme LLC, 2Department of Biomedical Engineering, University of Cincinnati, 3 The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro. 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. Biology Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes Perwez Alam1, Bryan D. Maliken1, Malina J. Ivey1, Shannon M. Jones1, Onur Kanisicak1 1Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes. Bioengineering Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification Zhenyuan Xu1, Jacob A. Orkwis1, Greg M. Harris1,2,3 1Department of Chemical and Environmental Engineering, University of Cincinnati, 2Department of Biomedical Engineering, University of Cincinnati, 3Neuroscience Graduate Program, University of Cincinnati College of Medicine This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype. Environment RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points Shubham Rathore1, Jenni Hassert1, Courtney M. Clark-Hachtel2,3, Aaron Stahl1,4, Yoshinori Tomoyasu2, Elke K. Bushbeck1 1Department of Biological Sciences, University of Cincinnati, 2Department of Biology, Miami University, 3Department of Biology, University of North Carolina at Chapel Hill, 4Department of Neuroscience, The Scripps Research Institute RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior. Immunology and Infection Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages Yuxuan Zhen1, Wen-Hai Shao1 1Division of Immunology, Allergy, and Rheumatology, Department of Internal Medicine, College of Medicine, University of Cincinnati Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria. Neuroscience A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury Brandon Foreman1, Daryn Cass2, Jonathan Forbes2, Laura B. Ngwenya2,3 1Department of Neurology and Rehabilitation Medicine, University of Cincinnati, 2Department of Neurosurgery, University of Cincinnati, 3Neurotrauma Center, University of Cincinnati Gardner Neuroscience Institute A method of recording multimodality monitoring signals in patients with severe brain injuries using a bedside, single burr hole technique is described. Developmental Biology Wholemount In Situ Hybridization for Astyanax Embryos Heidi Luc1, Connor Sears1, Andrew Raczka1, Joshua B. Gross1 1Department of Biological Sciences, University of Cincinnati This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining. Bioengineering Novel Process for 3D Printing Decellularized Matrices Stacey M. S. Gruber1, Paulomi Ghosh2, Karl Wilhelm Mueller2, Patrick W. Whitlock1,2,3, Chia-Ying Lin1,3 1Department of Biomedical Engineering, University of Cincinnati, 2 This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs. Medicine Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method Esam S.B. Salem*1,2, Kazutoshi Murakami*2, Toshimasa Takahashi2, Elise Bernhard2, Vishnupriya Borra2, Mridula Bethi2, Takahisa Nakamura2,3,4 1Department of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, 2 Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver. Medicine Collecting Hair Samples for Hair Cortisol Analysis in African Americans Kathy D. Wright1, Jodi L. Ford1, Joseph Perazzo2, Lenette M. Jones3, Sherrilynn Mahari4, Brent A. Sullenbarger5,6,7, Mark L. Laudenslager8 1College of Nursing, The Ohio State University, 2College of Nursing, University of Cincinnati, 3School of Nursing, University of Michigan, 4Licensed Cosmetologist Consultant, 5Clinical Research Manager, The Ohio State University, 6Biomedical Core Research Coordinator, The Ohio State University, 7College of Nursing Biomedical Core Research Laboratory, The Ohio State University, 8Department of Psychiatry, Behavioral Immunology & Endocrinology Laboratory, University of Colorado Denver Anschutz Medical Campus Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans. Immunology and Infection Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration Emma Teal1, Nina G. Steele2, Jayati Chakrabarti1, Loryn Holokai3, Yana Zavros1 1Department of Pharmacology and Systems Physiology, University of Cincinnati, 2Department of Cell & Developmental Biology, University of Michigan, 3Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described. Neuroscience Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition Michael D. Guthrie1, Donald L. Gilbert2, David A. Huddleston2, Ernest V. Pedapati2,3, Paul S. Horn2, Stewart H. Mostofsky4, Steve W. Wu2 1College of Medicine, University of Cincinnati, 2 We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task. Medicine Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease Victoria N. Jensen*1, Shannon H. Romer*2, Sarah M. Turner3, Steven A. Crone3 1Neuroscience Graduate Program, University of Cincinnati, 2Department of Biological Sciences, Wright State University, 3 This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device. Behavior Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors Jonathan L. Preston1,2, Tara McAllister Byun3, Suzanne E. Boyce2,4, Sarah Hamilton4, Mark Tiede2, Emily Phillips2, Ahmed Rivera-Campos4, Douglas H. Whalen2,5,6 1Department of Communication Sciences and Disorders, Syracuse University, 2Haskins Laboratories, 3Department of Communicative Sciences and Disorders, New York University, 4Department of Communication Sciences and Disorders, University of Cincinnati, 5Program in Speech-Language-Hearing Sciences, City University of New York Graduate Center, 6Department of Linguistics, Yale University Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations. Immunology and Infection Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens Swinburne A. J. Augustine1, Tarsha N. Eason2, Kaneatra J. Simmons1, Clarissa L. Curioso3, Shannon M. Griffin1, Malini K. D. Ramudit1, Trevor R. Plunkett4 1National Exposure Research Laboratory, U.S. Environmental Protection Agency, 2National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 3Oak Ridge Institute for Science and Education, 4Department of Biological Sciences, McMicken College of Arts and Sciences, University of Cincinnati In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously. Biology Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA) Daniel E. Miller1, Zubin H. Patel1,2,3, Xiaoming Lu1,3, Arthur T. Lynch1, Matthew T. Weirauch*1,4, Leah C. Kottyan*1 1Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding. Biology Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs) Johanna E. Kowalko1, Li Ma2, William R. Jeffery3 1Genetics, Development and Cell Biology, Iowa State University, 2Department of Biological Sciences, University of Cincinnati, 3Department of Biology, University of Maryland Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish. Medicine A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail Chun Yu Wong1,2,3, Margreet R. de Vries2,3, Yang Wang4, Joost R. van der Vorst3, Alexander L. Vahrmeijer3, Anton-Jan van Zonneveld1,2, Jaap F. Hamming3, Prabir Roy-Chaudhury4, Ton J. Rabelink1,2, Paul H. A. Quax2,3, Joris I. Rotmans1,2 1Department of Nephrology, Leiden University Medical Center, 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 3Department of Surgery, Leiden University Medical Center, 4Division of Nephrology, University of Cincinnati Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions. Medicine Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS Touraj Shokati1, Nicholas Bodenberger1, Holly Gadpaille1, Björn Schniedewind1, Alexander A. Vinks2, Wenlei Jiang3, Rita R. Alloway4, Uwe Christians1 1iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus, 2 Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction. Engineering Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers Nicholas Blumenschein1, Daewoo Han1, Andrew J. Steckl1 1Department of Electrical Engineering and Computing Systems, University of Cincinnati Here, we present a protocol to investigate multi-component phase diagrams using externally controlled magnetic beads as liquid carriers in a lab-in-tube approach. This approach can aid in applications that seek to gather further information on phase change in complex liquid systems. Behavior Vision Training Methods for Sports Concussion Mitigation and Management Joseph F. Clark1, Angelo Colosimo2, James K. Ellis3, Robert Mangine3, Benjamin Bixenmann4, Kimberly Hasselfeld2, Patricia Graman5, Hagar Elgendy2, Gregory Myer6, Jon Divine2 1Neurology and Rehabilitative Medicine, University of Cincinnati, 2Division of Sports Medicine, Department of Orthopaedic Surgery, University of Cincinnati, 3Department of Athletics, University of Cincinnati, 4Department of Neurosurgery, University of Cincinnati, 5College of Education, Criminal Justice, and Human Services, University of Cincinnati, 6 This paper describes a protocol to conduct, quantitatively monitor, and assess the success of vision training initiated as part of a sports medical management program including intervention for concussion prevention and performance enhancement. Neuroscience Olfactory Assays for Mouse Models of Neurodegenerative Disease Andrew M. Lehmkuhl1, Emily R. Dirr2,3, Sheila M. Fleming1,2 1Department of Psychology, University of Cincinnati, 2Department of Neurology, University of Cincinnati, 3Department of Neuroscience, Cell Biology, and Physiology, Wright State University Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases. Behavior Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease Sheila M. Fleming1,2, Osunde R. Ekhator1,2, Valentins Ghisays1 1Department of Psychology, University of Cincinnati, 2Department of Neurology, University of Cincinnati In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies. Biology Isolation and Primary Culture of Rat Hepatic Cells Ling Shen1, Allix Hillebrand2, David Q.-H. Wang3, Min Liu1 1Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 2American University in Washington, D.C., 3Department of Internal Medicine, Saint Louis University School of Medicine Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 × 108 cells per preparation with cell viability between 88 ~ 96%. Immunology and Infection Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient Sarah E. Staggs1, Mary Jean See2, J P. Dubey3, Eric N. Villegas2,4 1Dynamac, Inc., 2Department of Biological Sciences, University of Cincinnati, McMicken College of Arts and Science, 3Animal Parasitic Disease Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 4National Exposure Research Laboratory, US Environmental Protection Agency This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation