University of Louisville

23 articles published in JoVE

• Immunology and Infection

  
  Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis Jenna M. Olson¹, Jonathan M. Warawa^{1,2 1}Department of Microbiology and Immunology, University of Louisville, ²Center for Predictive Medicine, University of Louisville This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively.

• Cancer Research

  
  Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells Shuhan Meng^1,2, Aaron G. Whitt^1,2,3, Allison Tu², John W. Eaton^1,2,3, Chi Li^1,2,3, Kavitha Yaddanapudi^{4,5,6 1}Department of Pharmacology and Toxicology, University of Louisville, ²Experimental Therapeutics Program, Brown Cancer Center, University of Louisville, ³Department of Medicine, University of Louisville, ⁴Department of Surgery, University of Louisville, ⁵Immuno-Oncology Program, Brown Cancer Center, University of Louisville, ⁶Department of Microbiology and Immunology, University of Louisville This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells.

• Immunology and Infection

  
  Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line Katharina M. Scheurlen¹, Dylan L. Snook¹, Sarah A. Gardner¹, Maurice R. Eichenberger¹, Susan Galandiuk^{1 1}Price Institute of Surgical Research, Department of Surgery, University of Louisville M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

• Neuroscience

  
  Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds Lisa C. Ohman¹, Robin F. Krimm^{1 1}Anatomical Sciences and Neurobiology, University of Louisville This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

• Bioengineering

  
  Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells Connor S. Centner¹, Emily M. Murphy¹, Bryce F. Stamp², Mariah C. Priddy¹, John T. Moore¹, Paula J. Bates², Michael A. Menze³, Kavitha Yaddanapudi², Jonathan A. Kopechek^{1 1}Department of Bioengineering, University of Louisville, ²School of Medicine, University of Louisville, ³Department of Biology, University of Louisville This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

• Bioengineering

  
  Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair Takeichiro Nakane^1,2,6, Mosha Abulaiti^1,2, Yuko Sasaki¹, William J. Kowalski³, Bradley B. Keller^4,5,7, Hidetoshi Masumoto^{1,2 1}Clinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, ²Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, ³Laboratory of Stem Cell and Neuro-Vascular Biology, Cell and Developmental Biology Center, National Institutes of Health, ⁴Kosair Charities Pediatric Heart Research Program, Cardiovascular Innovation Institute, University of Louisville, ⁵Department of Pediatrics, School of Medicine, University of Louisville, ⁶Department of Cardiovascular Surgery, Mitsubishi Kyoto Hospital, ⁷ The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

• Medicine

  
  Slicing and Culturing Pig Hearts under Physiological Conditions Qinghui Ou*¹, Riham R.E. Abouleisa*¹, Xian-Liang Tang¹, Hamzah R. Juhardeen¹, Moustafa H. Meki², Jessica M. Miller², Guruprasad Giridharan², Ayman El-Baz², Roberto Bolli¹, Tamer M.A. Mohamed^{1,3,4,5,6 1}Institute of Molecular Cardiology, Department of Medicine, University of Louisville, ²Department of Bioengineering, University of Louisville, ³Diabetes and Obesity Center, Department of Medicine, University of Louisville, ⁴Department of Pharmacology and Toxicology, University of Louisville, ⁵Institute of Cardiovascular Sciences, University of Manchester, ⁶Faculty of Pharmacy, Zagazig University This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

• Developmental Biology

  
  Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography C. Ben Lovely^{1 1}Department of Biochemistry and Molecular Genetics, Alcohol Research Center, University of Louisville This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

• Medicine

  
  Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction Anna L. Lang^1,2, William T. Goldsmith^3,4, Regina D. Schnegelberger^5,6, Gavin E. Arteel^6,7, Juliane I. Beier^{6,7 1}Department of Pharmacology and Toxicology, University of Louisville, ²Hepatobiology and Toxicology Program, University of Louisville, ³Department of Physiology and Pharmacology, West Virginia University, ⁴Center for Inhalation Toxicology, West Virginia University, ⁵Department of Pharmacology and Chemical Biology, University of Pittsburgh, ⁶Pittsburgh Liver Research Center, University of Pittsburgh, ⁷Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

• Medicine

  
  Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats Natia Q. Kelm¹, Jason E. Beare^1,2, Amanda J. LeBlanc^{1,3 1}Cardiovascular Innovation Institute, University of Louisville, ²Kentucky Spinal Cord Injury Research Center, University of Louisville, ³Department of Physiology, University of Louisville Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

• Medicine

  
  Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research Yoram Fleissig¹, Robert M. Reed^2,3, Jason E. Beare^2,3, Amanda J. LeBlanc^2,4, Stuart K. Williams^2,4, Christina L. Kaufman¹, James B. Hoying^{2,4 1}Christine M. Kleinert Institute for Hand and Microsurgery, ²Cardiovascular Innovation Institute, University of Louisville, ³Kentucky Spinal Cord Injury Research Center, University of Louisville, ⁴Department of Physiology, University of Louisville Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

• Neuroscience

  
  Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats Jason H. Gumbel¹, Casey J. Steadman¹, Robert F. Hoey¹, James E. Armstrong¹, Jason D. Fell¹, Cui Bo Yang¹, Lynnette R. Montgomery¹, Charles H. Hubscher^{1,2 1}Department of Anatomical Sciences and Neurobiology, University of Louisville, ²Kentucky Spinal Cord Injury Rehabilitation Center, University of Louisville This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

• Bioengineering

  
  Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism Robert A. Skolik¹, Mary E. Konkle², Michael A. Menze^{1 1}Department of Biological Sciences, University of Louisville, ²Department of Chemistry, Ball State University This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

• Immunology and Infection

  
  Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death Yuwen Zhang¹, Jiaqing Hao¹, Yanwen Sun¹, Bing Li^{1 1}Department of Microbiology and Immunology, University of Louisville We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

• Bioengineering

  
  Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections Hung R. Vuong*¹, Kevin M. Tyo*^2,3, Jill M. Steinbach-Rankins^{2,3,4,5 1}Department of Chemistry, University of Louisville, ²Department of Pharmacology and Toxicology, University of Louisville, ³Center for Predictive Medicine, University of Louisville, ⁴Department of Microbiology and Immunology, University of Louisville, ⁵Department of Bioengineering, University of Louisville This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

• Medicine

  
  A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice Xiangbing Wu*¹, Yi Ping Zhang*², Wenrui Qu¹, Lisa B. E. Shields², Christopher B. Shields^2,3, Xiao-Ming Xu^{1 1}Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Indiana University School of Medicine, ²Norton Neuroscience Institute, Norton Healthcare, ³Department of Anatomical Sciences and Neurobiology, University of Louisville We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

• Engineering

  
  Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System Hanwen Yuan¹, Scott D. Cambron¹, Robert S. Keynton^{1 1}Bioengineering Department, University of Louisville Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and “web-like” structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

• Neuroscience

  
  An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time Starlyn L. M. Okada¹, Nicole S. Stivers¹, Peter K. Stys², David P. Stirling^{1 1}KY Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, ²Hotchkiss Brain Institute, Department of Clinical Neurosciences, University of Calgary We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

• Medicine

  
  Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury Sevda C. Aslan¹, Manpreet K. Chopra¹, William B. McKay², Rodney J. Folz³, Alexander V. Ovechkin^{1 1}Department of Neurological Surgery, University of Louisville, ²Spinal Cord Injury Laboratory, Shepherd Center, ³Department of Medicine: Division of Pulmonary, Critical Care and Sleep Disorders, University of Louisville The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

• Biology

  
  Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay Nazarul Hasan¹, David Humphrey¹, Krista Riggs¹, Chuan Hu^{1 1}Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.

• Medicine

  
  A Simplified Technique for Producing an Ischemic Wound Model Sufan Chien¹, Bradon J. Wilhelmi^{1 1}Department of Surgery, University of Louisville We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

• Immunology and Infection

  
  Real-time Imaging of Leukotriene B₄ Mediated Cell Migration and BLT1 Interactions with β-arrestin Venkatakrishna R. Jala¹, Bodduluri Haribabu^{1 1}Microbiology and Immunology, James Graham Brown Cancer Center, University of Louisville This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

• Biology

  
  Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells William N. White¹, Palaniappan Sethu^{2 1}Department of Mechanical Engineering, University of Louisville, ²Department of Bioengineering, University of Louisville An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.