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University of Tennessee Health Science Center

9 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex
Robert C. Foehring 1, Dongxu Guan 1, Tara Toleman 1, Angela R. Cantrell 1
1Department of Anatomy and Neurobiology, University of Tennessee Health Science Center

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

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Medicine

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain
Karl A. Sillay 1,2, S. Gray McClatchy 3, Brandon A. Shepherd 1, Garrett T. Venable 1, Tyler S. Fuehrer 4
1University of Tennessee Health Science Center, 2Semmes-Murphey Clinic, 3University of Arkansas for Medical Sciences, 4Restorative Neurosciences Foundation

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

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Bioengineering

Live Cell Imaging during Mechanical Stretch
Gabriel Rápalo 1,2, Josh D. Herwig 3, Robert Hewitt 4, Kristina R. Wilhelm 1,2, Christopher M. Waters 1,2, Esra Roan 3
1Department of Physiology, University of Tennessee Health Science Center, 2Department of Biomedical Engineering and Imaging, University of Tennessee Health Science Center, 3Department of Biomedical Engineering, University of Memphis, 4Department of Engineering Technology, University of Memphis

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

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Biology

Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice
Amali Samarasinghe 1,2, Scott A Hoselton 3, Jane M Schuh 3, Jonathan A McCullers 1,2
1Department of Pediatrics, University of Tennessee Health Science Center, 2Department of Infectious Diseases, St. Jude Children’s Research Hospital, 3Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo

Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host.

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Developmental Biology

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration
Chandrima Sinha *1,2, Kavisha Arora *1, Anjaparavanda P. Naren 1,2
1Division of Pulmonary Medicine, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, 2Department of Physiology, University of Tennessee Health Science Center

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

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Bioengineering

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry
Sumana R. Chintalapudi 1, Need N. Patel 1, Zachary K. Goldsmith 1, Levon Djenderedjian 1, Xiang Di Wang 1, Tony N. Marion 2, Monica M. Jablonski 1,3,4, Vanessa M. Morales-Tirado 1,2
1Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, 2Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 3Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, 4Department of Pharmaceutical Sciences, University of Tennessee Health Science Center

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

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Biochemistry

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis
Francisco J. Sierra-Valdez 1, Richard A. Stein 2, Phanindra Velissety 1,3, Valeria Vasquez 1, Julio F. Cordero-Morales 1
1Department of Physiology, University of Tennessee Health Science Center, 2Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 3CuriRX, Inc.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

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Biology

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

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

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JoVE Core

Disposable Dosators Intended for Dry Powder Delivery to Mice
Sara E. Maloney 1, Ian E. Stewart 1, Jeffrey B. Mecham 1, Mercedes Gonzalez-Juarrero 2, Bernd Meibohm 3, Anthony J. Hickey 1
1Technology Advancement and Commercialization, RTI International, 2Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

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