University of Tennessee Health Science Center View Institution's Website 11 articles published in JoVE Medicine Disposable Dosators Intended for Dry Powder Delivery to Mice Sara E. Maloney1, Ian E. Stewart1, Jeffrey B. Mecham1, Mercedes Gonzalez-Juarrero2, Bernd Meibohm3, Anthony J. Hickey1 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. Biology Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents John Malysz1, Eric S. Rovner2, Robert Wake3, Georgi V. Petkov1,3,4 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. Biochemistry Purification and Reconstitution of TRPV1 for Spectroscopic Analysis Francisco J. Sierra-Valdez1, Richard A. Stein2, Phanindra Velissety1,3, Valeria Vasquez1, Julio F. Cordero-Morales1 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. Bioengineering Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry Sumana R. Chintalapudi1, Need N. Patel1, Zachary K. Goldsmith1, Levon Djenderedjian1, Xiang Di Wang1, Tony N. Marion2, Monica M. Jablonski1,3,4, Vanessa M. Morales-Tirado1,2 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. Developmental Biology Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration Chandrima Sinha*1,2, Kavisha Arora*1, Anjaparavanda P. Naren1,2 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. Medicine Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry Mohammad Afaque Alam1, Cory Parks1, Salvatore Mancarella1 1Department of Physiology, University of Tennessee Health Sciences Center The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices. Bioengineering Live Cell Imaging during Mechanical Stretch Gabriel Rápalo1,2, Josh D. Herwig3, Robert Hewitt4, Kristina R. Wilhelm1,2, Christopher M. Waters1,2, Esra Roan3 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. Biology Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation Kathirvel Kandasamy1, Kaushik Parthasarathi1,2 1Department of Physiology, The University of Tennessee Health Science Center, 2Department of Orthopedic Surgery and Biomedical Engineering, The University of Tennessee Health Science Center The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging. Medicine Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain Karl A. Sillay1,2, S. Gray McClatchy3, Brandon A. Shepherd1, Garrett T. Venable1, Tyler S. Fuehrer4 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. Neuroscience Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis Joshua N. Douglas1,2,3, Lidia A. Gardner1,2, Sangmin Lee1,2, Yoojin Shin1,2, Chassidy J. Groover1,2, Michael C. Levin1,2,3 1Research Service, Veterans Administration Medical Center, Memphis, TN, 2Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, 3Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells. Neuroscience Whole Cell Recording from an Organotypic Slice Preparation of Neocortex Robert C. Foehring1, Dongxu Guan1, Tara Toleman1, Angela R. Cantrell1 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.
