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University of Kentucky

47 ARTICLES PUBLISHED IN JoVE

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Biology

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method
Alan Daugherty 1, Debra Rateri 1, Lu Hong 1, Anju Balakrishnan 1
1Cardiovascular Research Center, University of Kentucky

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

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Biology

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish
Sonya M. Bierbower 1, Robin L. Cooper 1
1Department of Biology, University of Kentucky

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

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Biology

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle
Ann S. Cooper 1, Robin L. Cooper 1
1Department of Biology, University of Kentucky

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

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Biology

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies
Ann S. Cooper 1, Kylah E. Rymond 1, Matthew A. Ward 1, Easter L. Bocook 1, Robin L. Cooper 1
1Department of Biology, University of Kentucky, Lexington

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

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Neuroscience

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle
Wen Hui Wu *1, Robin L. Cooper *1
1Department of Biology, University of Kentucky

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

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Neuroscience

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Brittany Baierlein *1, Alison L. Thurow *1, Harold L. Atwood *2, Robin L. Cooper *1
1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

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Neuroscience

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception
Bonnie Leksrisawat *1, Ann S. Cooper *1, Allison B. Gilberts *1, Robin L. Cooper *1
1Department of Biology, University of Kentucky

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

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Neuroscience

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise
Ann S. Cooper *1, Bonnie Leksrisawat *1, Allison B. Gilberts *1, A. Joffre Mercier *2, Robin L. Cooper *1
1Department of Biology, University of Kentucky, 2Department of Biological Sciences, Brock University

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

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Education

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises
Martha M. Robinson 1, Jonathan M. Martin 1, Harold L. Atwood 2, Robin L. Cooper 1
1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

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Biology

Mitochondrial Isolation from Skeletal Muscle
Mary L. Garcia-Cazarin *1, Natalie N. Snider *1, Francisco H. Andrade 1
1Department of Physiology, University of Kentucky

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

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Biology

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
Jessica S. Blackburn 1, Sali Liu 1, David M. Langenau 2
1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

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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

Neural Circuit Recording from an Intact Cockroach Nervous System
Josh S. Titlow 1, Zana R. Majeed 1,2, H. Bernard Hartman 3, Ellen Burns 1, Robin L. Cooper 1
1Department of Biology, University of Kentucky , 2Department of Biology, University of Salahaddin, 3Oregon Institute of Marine Biology, University of Oregon

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

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Neuroscience

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis
Josh Titlow 1, Zana R. Majeed 1,2, John G Nicholls 3, Robin L. Cooper 1
1Department of Biology, University of Kentucky, 2Department of Biology, College of Science, University of Salahaddin, Iraq, 3Department of Neurobiology and Cognitive Neuroscience, SISSA, Italy

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

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JoVE Journal

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises
Zana R. Majeed 1,2, Josh Titlow 1,2, H. Bernard Hartman 3, Robin Cooper 1,2
1Department of Biology, University of Kentucky, 2Center of Muscle Biology, University of Kentucky, 3Oregon Institute of Marine Biology, University of Oregon

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

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Biology

Tissue Triage and Freezing for Models of Skeletal Muscle Disease
Hui Meng 1, Paul M.L. Janssen 2, Robert W. Grange 3, Lin Yang 4, Alan H. Beggs 5, Lindsay C. Swanson 5, Stacy A. Cossette 1,6, Alison Frase 7, Martin K. Childers 8, Henk Granzier 9, Emanuela Gussoni 5, Michael W. Lawlor 1
1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

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Medicine

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice
Hong Lu 1, Deborah A. Howatt 1, Anju Balakrishnan 1, Jessica J. Moorleghen 1, Debra L. Rateri 1, Lisa A. Cassis 2, Alan Daugherty 1
1Saha Cardiovascular Research Center, University of Kentucky, 2Department of Pharmacology and Nutritional Sciences, University of Kentucky

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

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Biology

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking
Ashley M. Fox-Loe 1, Brandon J. Henderson 2, Christopher I. Richards 1
1Department of Chemistry, University of Kentucky, 2Department of Biomedical Sciences, Marshall University

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

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Developmental Biology

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles
Richard D. Andreatta 1, Joseph C. Stemple 1, Tanya S. Seward 2, Colleen A. McMullen 2
1Department of Rehabilitation Sciences, University of Kentucky, 2Department of Physiology, University of Kentucky

Here, we present a protocol to describe the use of neurotrophin 4 (NTF4) systemically and directly to remodel rat aging laryngeal muscles.

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Neuroscience

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue
Anika M.S. Hartz 1, Julia A. Schulz 2, Brent S. Sokola 2, Stephanie E. Edelmann 1, Andrew N. Shen 1, Ralf G. Rempe 2, Yu Zhong 1, Nader El Seblani 3, Bjoern Bauer 2
1Sanders-Brown Center on Aging, Department of Pharmacology and Nutritional Sciences, University of Kentucky, 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 3Department of Neuroscience, University of Kentucky

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

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Bioengineering

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana
B. K. Amos 1, Victoria G. Pook 1, Seth Debolt 1
1Department of Horticulture, University of Kentucky

A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

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Medicine

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans
Vedant A. Gupta 1, Steve W. Leung 1, Michael A. Winkler 2, Vincent L. Sorrell 1
1Gill Heart Institute, Division of Cardiovascular Medicine, University of Kentucky, 2Department of Radiology, University of Kentucky

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

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Environment

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar
Carrie A. Deans 1,2, Gregory A. Sword 1, Paul A. Lenhart 3, Eric Burkness 2, William D. Hutchison 2, Spencer T. Behmer 1
1Department of Entomology, Texas A&M University, 2Department of Entomology, University of Minnesota, 3Department of Entomology, University of Kentucky

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

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Biochemistry

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies
K. Martin Chow 1, Sidney W. Whiteheart 1, Jeffrey R. Smiley 2, Savita Sharma 1, Kathy Boaz 2, Meggie J. Coleman 2, Alvina Maynard 3, Louis B. Hersh 1, Craig W. Vander Kooi 1
1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Division of Laboratory Animal Resources, University of Kentucky, 3River Hill Ranch

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

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Biology

Bovine Mammary Gland Biopsy Techniques
Veridiana L. Daley 1,4, Charles Dye 2, Sophie H. Bogers 3, R. Michael Akers 4, Francisco C. Rodriguez 3, John P. Cant 5, John Doelman 5, Peter Yoder 4, Karan Kumar 2, Dane Webster 6, Mark D. Hanigan 4
1National Animal Nutrition Program, a National Research Support Project (NRSP-9), Department of Animal and Food Sciences, University of Kentucky, 2School of Performing Arts, Virginia Tech, 3Department of Large Animal Clinical Sciences, The Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 4Department of Dairy Science, Virginia Tech, 5Department of Animal Biosciences, University of Guelph, 6School of Visual Arts, Virginia Tech

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

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Bioengineering

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier
Wanzhu Zhao 1, Linjiang Han 1, Younsoo Bae 2, Devika S. Manickam 1
1Department of Pharmaceutical, Administrative and Social Sciences, Duquesne University, 2Department of Pharmaceutical Sciences, University of Kentucky

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

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JoVE Core

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes
Joseph S. Najem 1,2, Graham J. Taylor 2,3, Nick Armendarez 4, Ryan J. Weiss 5, Md Sakib Hasan 5, Garrett S. Rose 5, Catherine D. Schuman 6, Alex Belianinov 7, Stephen A. Sarles 2, C. Patrick Collier 2,3,7
1Joint Institute for Biological Sciences, Oak Ridge National Laboratory, 2Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, 3Bredesen Center for Interdisciplinary Research, University of Tennessee, 4Department of Biosystems and Agriculture Engineering, University of Kentucky, 5Department of Electrical Engineering and Computer Science, University of Tennessee, 6Computer Science and Mathematics Division, Oak Ridge National Laboratory, 7Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

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Medicine

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions
Hisashi Sawada 1, Jeff Z. Chen 2, Bradley C. Wright 1, Jessica J. Moorleghen 1, Hong S. Lu 1,2, Alan Daugherty 1,2
1Saha Cardiovascular Research Center, University of Kentucky, 2Department of Physiology, University of Kentucky

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

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Biochemistry

Use of Alu Element Containing Minigenes to Analyze Circular RNAs
Justin R. Welden 1, Anna Pawluchin 1, Jacob van Doorn 1, Stefan Stamm 1
1Institute for Molecular and Cellular Biochemistry, University of Kentucky

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

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Medicine

Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease
Jonathan H. Chung 1, Christopher M. Walker 2, Stephen Hobbs 3
1Department of Radiology, University of Chicago Medicine, 2Department of Radiology, University of Kansas Medical Center, 3Department of Radiology, University of Kentucky

Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease.

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Immunology and Infection

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis
Gabrielle Keb 1, Kenneth A. Fields 1
1Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

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Cancer Research

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish
Meghan G. Haney 1,2, L. Henry Moore 1, Jessica S. Blackburn 1,2
1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Markey Cancer Center, University of Kentucky

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

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Neuroscience

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia
Bei Zhang 1, Binoy Joseph 2, Kathryn E. Saatman 2, Lei Chen 2
1College of Public Health, Shaanxi University of Chinese Medicine, 2Spinal Cord and Brain Injury Research Center, Department of Physiology, University of Kentucky

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

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Biology

High-Throughput Assays of Critical Thermal Limits in Insects
David N. Awde 1, Tatum E. Fowler 1, Fernan Pérez-Gálvez 1, Mark J. Garcia 1, Nicholas M. Teets 1
1Department of Entomology, University of Kentucky

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

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Bioengineering

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells
Joree N. Sandin 1, Surya P. Aryal 2, Thomas Wilkop 3,4, Christopher I. Richards 2,4, Martha E. Grady 1
1Department of Mechanical Engineering, University of Kentucky, 2Department of Chemistry, University of Kentucky, 3Department of Physiology, University of Kentucky, 4UK Light Microscopy Core, University of Kentucky

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

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Environment

Simulating Impacts of Ice Storms on Forest Ecosystems
John L. Campbell 1, Lindsey E. Rustad 1, Charles T. Driscoll 2, Ian Halm 3, Timothy J. Fahey 4, Habibollah Fakhraei 5, Peter M. Groffman 6,7, Gary J. Hawley 8, Wendy Leuenberger 9, Paul G. Schaberg 10
1Northern Research Station, U.S. Forest Service, Durham, NH, 2Department of Civil and Environmental Engineering, Syracuse University, 3Northern Research Station, U.S. Forest Service, North Woodstock, NH, 4Department of Natural Resources, Cornell University, 5Department of Civil and Environmental Engineering, Southern Illinois University, 6Advanced Science Research Center at the Graduate Center, City University of New York, 7Cary Institute of Ecosystem Studies, 8Rubenstein School of Environment and Natural Resources, University of Vermont, 9Department of Forestry and Natural Resources, University of Kentucky, 10Northern Research Station, U.S. Forest Service, Burlington, VT

Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

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Neuroscience

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells
Carolina Galeano-Naranjo 1,2, A. Catalina Veléz-Ortega 1, Gregory I Frolenkov 1
1Department of Physiology, College of Medicine, University of Kentucky, 2Universidad Nacional de Colombia

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

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Biology

In vivo Measurement of Knee Extensor Muscle Function in Mice
Camille R. Brightwell 1,2, Ted G. Graber 3, Benjamin D. Brightwell 4,5, Matthew Borkowski 6, Brian Noehren 5,7, Christopher S. Fry 1,2
1Department of Athletic Training and Clinical Nutrition, University of Kentucky, 2Center for Muscle Biology, University of Kentucky, 3Department of Physical Therapy, East Carolina University, 4Kinesiology and Health Promotion Graduate Program, University of Kentucky, 5Biomotion Lab, College of Health Sciences, University of Kentucky, 6Aurora Scientific, 7Department of Physical Therapy, College of Health Sciences, University of Kentucky

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

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Developmental Biology

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish
Rebecca A. Petersen 1, Ann C. Morris 1
1Department of Biology, University of Kentucky

Here, a protocol is provided for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. 

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Biology

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda
Xien Chen 1, Subba Reddy Palli 1
1Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

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Bioengineering

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording
Julianna M. Richie 1, Paras R. Patel 1, Elissa J. Welle 1, Tianshu Dong 2, Lei Chen 3, Albert J. Shih 2, Cynthia A. Chestek 1,4,5,6
1Department of Biomedical Engineering, University of Michigan, Ann Arbor, 2Department of Mechanical Engineering, University of Michigan, Ann Arbor, 3Department of Mechanical Engineering, University of Massachusetts Lowell, 4Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, 5Neuroscience Graduate Program, University of Michigan, Ann Arbor, 6Robotics Graduate Program, University of Michigan, Ann Arbor

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

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Neuroscience

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin
Brandon A. Miller 1,2, Shelei Pan 3, Peter H. Yang 3, Catherine Wang 1, Amanda L. Trout 1, Dakota DeFreitas 3, Sruthi Ramagiri 3, Scott D. Olson 2, Jennifer M. Strahle 3,4,5
1Department of Neurosurgery, University of Kentucky, 2Department of Pediatric Surgery, University of Texas, 3Department of Neurological Surgery, Washington University in St. Louis School of Medicine, 4Department of Orthopedic Surgery, Washington University in St. Louis School of Medicine, 5Department of Pediatrics, Washington University in St. Louis School of Medicine

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

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Neuroscience

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice
Teresa Macheda 1, Kelly Roberts 1, Adam D. Bachstetter 1,2,3
1Spinal Cord & Brain Injury Research Center, University of Kentucky, 2Department of Neuroscience, University of Kentucky, 3Sanders-Brown Center on Aging, University of Kentucky

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

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Biology

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome
Bilin Liu 1,2, Anqi Li 1, Yuan Qin 3, Lei Chen 1, Meng Gao 1, Guohua Gong 1,2
1Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, 2Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 3Department of Pharmacy, Shanghai East Hospital, Tongji University

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

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Bioengineering

Equipment Setup and Artifact Removal for Simultaneous Electroencephalogram and Functional Magnetic Resonance Imaging for Clinical Review in Epilepsy
Jihye Bae 1, Jordan L. Clay 2, Bhoj Raj Thapa 1, David Powell 3, Heidi Turpin 3, Saghi Tasori Partovi 3, Rachel Ward-Mitchell 3, Balu Krishnan 4, Andreas Koupparis 5, Meriem Bensalem Owen 2, Flavius D. Raslau 2,6,7
1Department of Electrical and Computer Engineering, University of Kentucky, 2Department of Neurology, University of Kentucky, 3Department of Neuroscience, University of Kentucky, 4Cleveland Clinic, 5Cyprus Institute of Neurology and Genetics, 6Department of Radiology, University of Kentucky, 7Department of Neurosurgery, University of Kentucky

This article details simultaneous electroencephalogram and functional magnetic resonance imaging (EEG-fMRI) recording procedures that can be used in both clinical and research settings. EEG processing procedures to remove imaging artifacts for clinical review are also included. This study focuses on the example of epilepsy during the interictal period.

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Medicine

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis
Smita Joshi *1, Alexis N. Smith 1, Kanakanagavalli Shravani Prakhya 1, Hammodah R. Alfar 1, Joshua Lykins 1, Ming Zhang 1, Irina Pokrovskaya 2, Maria Aronova 3, Richard D. Leapman 3, Brian Storrie 2, Sidney W. Whiteheart *1
1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, 3Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

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Biology

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens
Renata Belisário 1, Maria F. Torres 1,2, Ester A. S. Buiate 1,3, Katia V. Xavier 1,4, Etta M. Nuckles 1, Lisa J. Vaillancourt 1
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.

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