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University of Louisville

31 ARTICLES PUBLISHED IN JoVE

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Biology

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells
William N. White 1, Palaniappan Sethu 2
1Department 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.

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Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin
Venkatakrishna R. Jala 1, Bodduluri Haribabu 1
1Microbiology 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.

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Medicine

A Simplified Technique for Producing an Ischemic Wound Model
Sufan Chien 1, Bradon J. Wilhelmi 1
1Department 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.

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Engineering

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation
Jonathan A. Kopechek 1, Peng Zhang 1, Mark T. Burgess 1, Tyrone M. Porter 1
1Department of Mechanical Engineering, Boston University

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

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Medicine

Controlled Cervical Laceration Injury in Mice
Yi Ping Zhang 1, Melissa J. Walker 2, Lisa B. E. Shields 1, Xiaofei Wang 2, Chandler L. Walker 2, Xiao-Ming Xu 2, Christopher B. Shields 1
1Norton Neuroscience Institute, Norton Healthcare, 2Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Medical Neuroscience Graduate Program, and Department of Anatomy and Cell Biology, Indiana University School of Medicine

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm.

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Medicine

A Novel Vertebral Stabilization Method for Producing Contusive Spinal Cord Injury
Melissa J. Walker *1,2, Chandler L. Walker *1,3, Y. Ping Zhang 4, Lisa B. E. Shields 4, Christopher B. Shields 4, Xiao-Ming Xu 1,3
1Spinal 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, 2Medical Neuroscience Graduate Program, Indiana University School of Medicine, 3Department of Anatomy and Cell Biology, Indiana University School of Medicine, 4Norton Neuroscience Institute, Norton Healthcare

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.  Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

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Medicine

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury
Sevda C. Aslan 1, Manpreet K. Chopra 1, William B. McKay 2, Rodney J. Folz 3, Alexander V. Ovechkin 1
1Department 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.

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Biology

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy
Patrick D. McGurk 1, C. Ben Lovely 1, Johann K. Eberhart 1
1Institute for Cell and Molecular Biology, The University of Texas at Austin

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

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Neuroscience

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
Starlyn L. M. Okada 1, Nicole S. Stivers 1, Peter K. Stys 2, David P. Stirling 1
1KY 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.

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Engineering

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System
Hanwen Yuan 1, Scott D. Cambron 1, Robert S. Keynton 1
1Bioengineering 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.

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Medicine

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice
Xiangbing Wu *1, Yi Ping Zhang *2, Wenrui Qu 1, Lisa B. E. Shields 2, Christopher B. Shields 2,3, Xiao-Ming Xu 1
1Spinal 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.

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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
1Department 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.

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Immunology and Infection

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death
Yuwen Zhang 1, Jiaqing Hao 1, Yanwen Sun 1, Bing Li 1
1Department 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.

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Neuroscience

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury
Chen Chen 1,2, Yi Ping Zhang 3, Yan Sun 1,4, Wenhui Xiong 1, Lisa B. E. Shields 3, Christopher B. Shields 3,5, Xiaoming Jin 1, Xiao-Ming Xu 1
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, and Department of Neurological Surgery, Indiana University School of Medicine, 2Program in Medical Neuroscience, Stark Neurosciences Research Institute, Indiana University School of Medicine, 3Norton Neuroscience Institute, Norton Healthcare, 4Department of Human Anatomy & Histoembryology, School of Basic Medical Sciences, Fudan University, 5Department of Neurological Surgery, University of Louisville School of Medicine

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

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Bioengineering

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism
Robert A. Skolik 1, Mary E. Konkle 2, Michael A. Menze 1
1Department 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.

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Genetics

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy
Jessica M. Miller *1, Nikhil Maneesh Mardhekar *1, Vasanthi Rajasekaran 1, Jianyi Zhang 1, Ram Kannappan 1
1Department of Biomedical Engineering, School of Engineering, School of Medicine, University of Alabama at Birmingham

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

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Neuroscience

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats
Jason H. Gumbel 1, Casey J. Steadman 1, Robert F. Hoey 1, James E. Armstrong 1, Jason D. Fell 1, Cui Bo Yang 1, Lynnette R. Montgomery 1, Charles H. Hubscher 1,2
1Department 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.

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Medicine

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats
Natia Q. Kelm 1, Jason E. Beare 1,2, Amanda J. LeBlanc 1,3
1Cardiovascular 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.

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Medicine

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research
Yoram Fleissig 1, Robert M. Reed 2,3, Jason E. Beare 2,3, Amanda J. LeBlanc 2,4, Stuart K. Williams 2,4, Christina L. Kaufman 1, James B. Hoying 2,4
1Christine 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.

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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 Tu 2, John W. Eaton 1,2,3, Chi Li 1,2,3, Kavitha Yaddanapudi 4,5,6
1Department 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.

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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
1Department 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.

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Developmental Biology

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography
C. Ben Lovely 1
1Department 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.

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Medicine

Slicing and Culturing Pig Hearts under Physiological Conditions
Qinghui Ou *1, Riham R.E. Abouleisa *1, Xian-Liang Tang 1, Hamzah R. Juhardeen 1, Moustafa H. Meki 2, Jessica M. Miller 2, Guruprasad Giridharan 2, Ayman El-Baz 2, Roberto Bolli 1, Tamer M.A. Mohamed 1,3,4,5,6
1Institute 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.

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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 Sasaki 1, William J. Kowalski 3, Bradley B. Keller 4,5,7, Hidetoshi Masumoto 1,2
1Clinical 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, 7Cincinnati Children's Heart Institute

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.

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Bioengineering

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells
Connor S. Centner 1, Emily M. Murphy 1, Bryce F. Stamp 2, Mariah C. Priddy 1, John T. Moore 1, Paula J. Bates 2, Michael A. Menze 3, Kavitha Yaddanapudi 2, Jonathan A. Kopechek 1
1Department 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.

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Neuroscience

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds
Lisa C. Ohman 1, Robin F. Krimm 1
1Anatomical 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.

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Immunology and Infection

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Katharina M. Scheurlen 1, Dylan L. Snook 1, Sarah A. Gardner 1, Maurice R. Eichenberger 1, Susan Galandiuk 1
1Price 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.

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Immunology and Infection

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis
Jenna M. Olson 1, Jonathan M. Warawa 1,2
1Department 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.

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Developmental Biology

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens
Liang Bao 1, Mark P. Running 1
1Department of Biology, University of Louisville

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

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Developmental Biology

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
John R. Klem 1, Raèden Gray 1, C. Ben Lovely 1
1Alcohol Research Center, Department of Biochemistry and Molecular Genetics, University of Louisville

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

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Chemistry

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy
Johann V. Hemmer *1, Padmanabh B. Joshi *1, Andrew J. Wilson 1
1Department of Chemistry, University of Louisville

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

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