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louisiana state university health sciences center

26 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin
Travis M. Doggett 1, Jerome W. Breslin 1
1Department of Physiology, Louisiana State University Health Sciences Center

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

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Immunology and Infection

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis
Junko Yano 1, Paul L. Fidel, Jr. 1
1Louisiana State University Health Sciences Center

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

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Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics
Flavia M. Souza-Smith 1, Kristine M. Kurtz 1, Jerome W. Breslin 1
1Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

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Biology

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy
Prachi Jain 1, Rebecca A. Worthylake 2, Suresh K. Alahari 1,3
1Department of Biochemistry and Molecular Biology, LSU School of Medicine, 2Department of Oral Biology, LSU School of Dentistry, 3Stanley S. Scott Cancer Center, LSU School of Medicine

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

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Neuroscience

Methods to Assay Drosophila Behavior
Charles D. Nichols 1, Jaime Becnel 1, Udai B. Pandey 2
1Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 2Department of Genetics, Louisiana State University Health Sciences Center

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies
Wei Li 1, Shu Zhu 1, Yusong Zhang 1, Jianhua Li 1, Andrew E. Sama 1, Ping Wang 1, Haichao Wang 1
1The Feinstein Institute for Medical Research, North Shore – LIJ Health System

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

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Neuroscience

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol
Marco Pacifici 1,2, Francesca Peruzzi 1,2
1LSU Health Sciences Center - New Orleans, 2Medical School and Stanley S. Scott Cancer Center

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

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Immunology and Infection

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay
Maciej T. Nogalski 1,2, Gary C.T. Chan 3, Emily V. Stevenson 1,2, Donna K. Collins-McMillen 1,2, Andrew D. Yurochko 1,2,4
1Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 2Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, 3Department of Microbiology and Immunology, SUNY Upstate Medical University, 4Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

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Neuroscience

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area
Michael F. Salvatore 1, Brandon S. Pruett 1, Charles Dempsey 1, Victoria Fields 1
1Department of Pharmacology, Toxicology, & Neuroscience, Louisiana State University Health Sciences Center

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

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Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)
Xiaolin Tian 1, Mingwei Zhu 1, Long Li 1, Chunlai Wu 1
1Neuroscience Center of Excellence, Louisiana State University Health Sciences Center

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

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Medicine

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice
Norman R. Harris 1, Megan N. Watts 1, Wendy Leskova 1
1Department of Molecular & Cellular Physiology, Louisiana State University Health Sciences Center

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

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Medicine

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR
Marco Pacifici 1, Serena Delbue 2, Ferdous Kadri 1, Francesca Peruzzi 1
1Medical School and Stanley S. Scott Cancer Center, LSU Health Sciences Center, 2Department of Biomedical, Surgery and Dental Sciences, University of Milan

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

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Behavior

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Jennifer C. Arnold 1, Michael F. Salvatore 1
1Department of Pharmacology, Toxicology, & Neuroscience, Louisiana State University Health Sciences Center

Exercise may alleviate behavioral impairments associated with neurodegenerative diseases. Studies of the mechanisms underlying exercise-mediated effects have increased in disease models, but the neurobiological background accompanying these diseases, specifically aging, is likely not considered. Here, we illustrate measures to increase the likelihood of a successful exercise paradigm in aged rats.

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Medicine

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis
Shengli Dong *1, Mazvita Maziveyi *1, Suresh K. Alahari 1
1Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

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Medicine

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction
Renjith Parameswaran Nair 1, Changyu Zheng 2, Gulshan Sunavala-Dossabhoy 1
1Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 2Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, NIH, DHHS

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

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Biology

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress
Luke A. White 1, Emily V. Stevenson 1, J. Winny Yun 1, Randa Eshaq 1, Norman R. Harris 1, David K. Mills 2, Alireza Minagar 3, Pierre-Olivier Couraud 4, J. Steven Alexander 1
1Molecular and Cellular Physiology, Louisiana State University Health Sciences Center in Shreveport, 2Biological Sciences, Louisiana Tech University, 3Neurology, Louisiana State University Health Sciences Center in Shreveport, 4Institut Cochin, Inserm U1016, Cnrs Umr8104, Université Paris Descartes

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

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JoVE Journal

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory
Thea M. Edwards 1,2, Howard E. Morgan 2,3, Coralia Balasca 4, Naveen K. Chalasani 5, Lauren Yam 4,6, Alison M. Roark 4
1Department of Biology, University of the South, 2School of Biological Sciences, Louisiana Tech University, 3School of Medicine, Louisiana State University Health Sciences Center, 4Department of Biology, Furman University, 5Department of Computer Science, Louisiana Tech University, 6Clemson University

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ERα) and/or beta (ERβ). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

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Neuroscience

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction
Mychal S. Grames 1, Kasey L. Jackson 1, Robert D. Dayton 1, John A. Stanford 2, Ronald L. Klein 1
1Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, 2Department of Molecular & Integrative Physiology, University of Kansas Medical Center

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

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Genetics

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy
Vikas Mishra *1, Nicole M. Gautier *1, Edward Glasscock 1
1Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

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Behavior

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue
Steven D. Scahill 1, Maxwell Hunt 2, Camille L. Rogers 2, Frank H. Lau 2
1Department of Pharmacology, Louisiana State University Health Sciences Center, 2Department of Surgery, Louisiana State University Health Sciences Center

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

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Biochemistry

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR
Alex Kirui *1, Malitha C. Dickwella Widanage *1, Frederic Mentink-Vigier 2, Ping Wang 3, Xue Kang 1, Tuo Wang 1
1Department of Chemistry, Louisiana State University, 2National High Magnetic Field Laboratory, 3Departments of Pediatrics, and Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

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Immunology and Infection

Isolation, Transfection, and Culture of Primary Human Monocytes
Karlie Plaisance-Bonstaff 1,2, Celeste Faia 2, Dorota Wyczechowska 1, Duane Jeansonne 1, Cecilia Vittori 1,3, Francesca Peruzzi 1,2
1Department of Medicine, Louisiana State University Health Sciences Center, 2Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, 3Department of Biomedical and Clinical Sciences L. Sacco, University of Milan

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

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Biology

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues
Chang Liu 1, Shuangshuang Wang 1, Man Liu 2, Fuxiang Bai 1, Zhihong Chen 3, Ping Wang 4, Jun Mi 1,5, Xunwei Wu 1
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Shijiazhuang Shimen Experimental School, 3Qilu Children's Hospital of Shandong University, 4Qilu Hospital of Shandong University, 5Shenzhen Research Institute of Shandong University

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

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Immunology and Infection

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model
Junko Yano 1, Elizabeth A. Lilly 2, Mairi C. Noverr 2, Paul L. Fidel 1
1Center for Oral and Craniofacial Biology, Louisiana State University Health-School of Dentistry, 2Department of Microbiology and Immunology, Tulane University School of Medicine

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

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Cancer Research

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System
Loren M. Brown 1, Katherine L. Hebert 2, Rakesh R. Gurrala 3, C. Ethan Byrne 4, Matthew Burow 5, Elizabeth C. Martin 4, Frank H. Lau 1
1Department of Surgery, Louisiana State University Health Sciences Center, 2Department of Bioinnovation, Tulane University, 3Tulane University School of Medicine, 4Department of Biological and Agricultural Engineering, Louisiana State University, 5Department of Medicine, Tulane University School of Medicine

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

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Medicine

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice
Praveen Kumar 1, Man Si 1, Kelsey Paulhus 1, Edward Glasscock 1
1Department of Biological Sciences, Southern Methodist University

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

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