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72 ARTICLES PUBLISHED IN JoVE

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Biology

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes
Sunny Shah 1, Alexander Revzin 1
1Department of Biomedical Engineering, University of California, Davis

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

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Biology

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue
Meirigeng Qi 1, Barbara Barbaro 1, Shusen Wang 1, Yong Wang 1, Mike Hansen 1, Jose Oberholzer 1
1Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

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Biology

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets
Meirigeng Qi 1, Barbara Barbaro 1, Shusen Wang 1, Yong Wang 1, Mike Hansen 1, Jose Oberholzer 1
1Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

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Biology

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device
Adeola F. Adewola 1, Yong Wang 1, Tricia Harvat 1, David T. Eddington 2, Dongyoung Lee 1, Jose Oberholzer 1,2
1Department of Surgery, University of Illinois, Chicago, 2Department of Bioengineering, University of Illinois, Chicago

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

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Biology

IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Tessa R. Davis 1, Adam G. Schrum 1
1Department of Immunology, College of Medicine, Mayo Clinic

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

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Biology

Immunostaining of Dissected Zebrafish Embryonic Heart
Jingchun Yang 1, Xiaolei Xu 1
1Division of Cardiovascular Diseases, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

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Medicine

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test
Christopher T. Creedon 1, Pieter-Jan Verhulst 1, Kyoung M. Choi 1, Jessica E. Mason 1, David R. Linden 1, Joseph H. Szurszewski 1, Simon J. Gibbons 1, Gianrico Farrugia 1
1Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

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Bioengineering

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level
Joe Fu-Jiou Lo 1, Yong Wang 2,3, Zidong Li 1, Zhengtuo Zhao 1, Di Hu 1, David T. Eddington 3, Jose Oberholzer 2,3
1Department of Mechanical Engineering, University of Michigan-Dearborn, 2Department of Surgery/Transplant, University of Illinois at Chicago, 3Department of Bioengineering, University of Illinois at Chicago

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

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Bioengineering

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method
Nathan J. Wittenberg 1, Timothy W. Johnson 1, Luke R. Jordan 2, Xiaohua Xu 3, Arthur E. Warrington 3, Moses Rodriguez 3,4, Sang-Hyun Oh 1,2
1Department of Electrical and Computer Engineering, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Neurology, Mayo Clinic College of Medicine, 4Department of Immunology, Mayo Clinic College of Medicine

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

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Biology

Tissue Triage and Freezing for Models of Skeletal Muscle Disease
Hui Meng 1, Paul M.L. Janssen 2, Robert W. Grange 3, Lin Yang 4, Alan H. Beggs 5, Lindsay C. Swanson 5, Stacy A. Cossette 1,6, Alison Frase 7, Martin K. Childers 8, Henk Granzier 9, Emanuela Gussoni 5, Michael W. Lawlor 1
1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

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Biology

Fluorescence Imaging with One-nanometer Accuracy (FIONA)
Yong Wang *1,2, En Cai *1,2, Janet Sheung 1,2, Sang Hak Lee 1,2, Kai Wen Teng 2,3, Paul R. Selvin 1,2,3
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

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Medicine

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions
Sofia Karkampouna 1, Peter Kloen 2, Miryam C. Obdeijn 3, Scott M. Riester 4, Andre J. van Wijnen 4,5, Marianna Kruithof-de Julio 1,6
1Department of Molecular Cell Biology, Cancer Genomics Centre and Centre for Biomedical Genetics, Leiden University Medical Center, 2Department of Orthopedic Surgery, Academic Medical Center, 3Department of Plastic, Reconstructive and Hand Surgery, Academic Medical Center, 4Department of Orthopedic Surgery, Mayo Clinic, 5Department of Biochemistry and Molecular Biology, Mayo Clinic, 6Department of Dermatology, Leiden University Medical Center

Dupuytren’s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

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JoVE Journal

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis
Choong Yong Ung 1, Feng Guo 2, Xiaoling Zhang 3, Zhihui Zhu 3, Shizhen Zhu 1,3
1Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic College of Medicine, Center for Individualized Medicine, 2Tufts University School of Medicine, 3Department of Biochemistry and Molecular Biology, Mayo Clinic

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

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Medicine

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring
Maria V. Irazabal 1, Prasanna K. Mishra 2, Vicente E. Torres 1, Slobodan I. Macura 2
1Department of Internal Medicine, Division of Nephrology, Mayo Clinic, 2Department of Biochemistry and Molecular Biology, Mayo Clinic

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

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Medicine

Reduction of Iatrogenic Atrial Septal Defects with an Anterior and Inferior Transseptal Puncture Site when Operating the Cryoballoon Ablation Catheter
Michael E. Rich 1, Andrew Tseng 2, Hae W. Lim 3, Paul J. Wang 4, Wilber W. Su 1
1Department of Cardiovascular Medicine, Cavanagh Heart Center, Banner-University Medical Center, 2Mayo Medical School, Mayo Clinic, 3AF Solutions, Medtronic plc, 4Cardiology Division, Stanford University School of Medicine, Stanford University

The goal of this study is to demonstrate the preferential location of transseptal puncture during a cryoballoon catheter ablation procedure for the treatment of atrial fibrillation.

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Bioengineering

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles
Brandon J. Tefft 1, Susheil Uthamaraj 2, J. Jonathan Harburn 3, Martin Klabusay 4, Dan Dragomir-Daescu 2,5, Gurpreet S. Sandhu 1
1Division of Cardiovascular Diseases, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3School of Medicine, Pharmacy and Health, Durham University, 4Regional Center for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 5Mayo Clinic College of Medicine, Mayo Clinic

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

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Bioengineering

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention
Susheil Uthamaraj 1, Brandon J. Tefft 2, Ota Hlinomaz 3, Gurpreet S. Sandhu 2, Dan Dragomir-Daescu 1,4
1Division of Engineering, Mayo Clinic, 2Division of Cardiovascular Diseases, Mayo Clinic, 3Department of Cardioangiology, ICRC, St. Anne's University Hospital, 4Mayo Clinic College of Medicine

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

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Medicine

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice
Grace Casaclang-Verzosa 1, Maurice Enriquez-Sarano 2, Hector R. Villaraga 2, Jordan D. Miller 1,3
1Department of Surgery, Mayo Clinic, 2Department of Medicine, Mayo Clinic, 3Department of Physiology & Biomedical Engineering, Mayo Clinic

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

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Immunology and Infection

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study
Jens O. Watzlawik 1,2,3, Robert J. Kahoud 1,3,5, Bharath Wootla 1,2,3, Meghan M. Painter 1,2,3, Arthur E. Warrington 1,2,3, William A. Carey 3,4, Moses Rodriguez 1,2,3
1Department of Neurology, Mayo Clinic, 2Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic, 3Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic, 4Division of Neonatal Medicine, Mayo Clinic, 5Department of Pediatric and Adolescent Medicine, Mayo Clinic

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (Vκ) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

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Medicine

Intravascular Delivery of Biologics to the Rat Kidney
Federico Franchi 1, Xiang Yang Zhu 2, Tyra A. Witt 1, Lilach O. Lerman 2, Martin Rodriguez-Porcel 1
1Divisions of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, 2Divisions of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

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Medicine

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents
Susheil Uthamaraj 1, Brandon J. Tefft 2, Soumen Jana 2, Ota Hlinomaz 3, Manju Kalra 4, Amir Lerman 2, Dan Dragomir-Daescu 1,5, Gurpreet S. Sandhu 2
1Division of Engineering, Mayo Clinic, 2Department of Cardiovascular Diseases, Mayo Clinic, 3Department of Cardioangiology, ICRC, St. Anne's University Hospital, 4Department of Vascular Surgery, Mayo Clinic, 5Department of Physiology and Biomedical Engineering, Mayo Clinic

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

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Medicine

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis
Dan Dragomir-Daescu 1,2, Asghar Rezaei 1,2, Susheil Uthamaraj 2, Timothy Rossman 2, James T. Bronk 3, Mark Bolander 3, Vincent Lambert 2, Sean McEligot 2, Rachel Entwistle 2, Hugo Giambini 3, Iwona Jasiuk 4, Michael J. Yaszemski 3, Lichun Lu 1,3
1Department of Physiology and Biomedical Engineering, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3Department of Orthopedic Surgery, Mayo Clinic, 4Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

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JoVE Core

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position
Dan Dragomir-Daescu 1,2, Asghar Rezaei 1,2, Timothy Rossman 2, Susheil Uthamaraj 2, Rachel Entwistle 2, Sean McEligot 2, Vincent Lambert 2, Hugo Giambini 3, Iwona Jasiuk 4, Michael J. Yaszemski 3, Lichun Lu 1,3
1Department of Physiology and Biomedical Engineering, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3Department of Orthopedic Surgery, Mayo Clinic, 4Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

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Bioengineering

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation
Timothy Rossman 1, Susheil Uthamaraj 1, Asghar Rezaei 1,2, Sean McEligot 1, Hugo Giambini 3, Iwona Jasiuk 4, Michael J. Yaszemski 3, Lichun Lu 3, Dan Dragomir-Daescu 1,2
1Division of Engineering, Mayo Clinic, 2Department of Physiology and Biomedical Engineering, Mayo Clinic, 3Department of Orthopedic Surgery, Mayo Clinic, 4Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

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Medicine

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model
Dimitra Kotsougiani 1, Caroline A. Hundepool 1, Joost I. Willems 1, Patricia Friedrich 1, Alexander Y. Shin 1, Allen T. Bishop 1
1Microvascular Research Laboratory, Department of Orthopedic Surgery, Mayo Clinic

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

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Developmental Biology

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy
Manuela Lahne 1, Ryne A. Gorsuch 1, Craig M. Nelson 1,2, David R. Hyde 1
1Department of Biological Sciences, University of Notre Dame, 2Department of Neurosurgery, Mayo Clinic

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

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Synthesis and Characterization of Charged Hydrogels for the Extended Delivery of Vancomycin
Carl T. Gustafson 1, Felix Boakye-Agyeman 2, Cassandra L. Brinkman 3, Joel M. Reid 1, Robin Patel 3, Zeljko Bajzer 4,5, Mahrokh Dadsetan 5, Michael J. Yaszemski 5,6,1
1Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School, Mayo Clinic College of Medicine, Mayo Clinic, 2Pharmacometrics Center, Duke Clinical Research Institute, 3Department of Laboratory Medicine and Pathology, Mayo Clinic, 4Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, 5Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Mayo Clinic, 6Department of Orthopedic Surgery, Mayo Graduate School, Mayo Clinic College of Medicine, Mayo Clinic

Here we describe the synthesis and use of oligo(poly(ethylene glycol)fumarate) / sodium methacrylate (OPF/SMA) charged copolymers as an affinity based delivery system for vancomycin.

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Biology

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator
Adam J. Rossano 1, Michael F. Romero 1,2
1Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 2Department of Nephrology and Hypertension, Mayo Clinic College of Medicine

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

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Biology

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter
Krista L Thompson 1, Thomas R Meier 1, Jodi A Scholz 1
1Department of Comparative Medicine, Mayo Clinic

Endotracheal intubation in rabbits is challenging due to their unusual anatomy. Here we present a technique for direct intubation of the trachea using a polypropylene catheter as a guide. This method utilizes relatively inexpensive supplies, requires minimal training, and can be easily performed in any clinical setting.

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Neuroscience

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine
Mark D. Unger 1, Timothy P. Maus 2, Ross C. Puffer 3, Laura K. Newman 1, Bradford L. Currier 4, Andreas S. Beutler 1
1Departments of Anesthesiology and Oncology, Mayo Clinic, Translational Science Track, Mayo Graduate School, 2Department of Radiology (Section of Interventional Pain Management), Mayo Clinic, 3Department of Neurologic Surgery, Mayo Clinic, 4Department of Orthopedic Surgery, Mayo Clinic

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

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Immunology and Infection

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion
James J. Faust 1,2, Wayne Christenson 3,4,5, Kyle Doudrick 6, John Heddleston 7, Teng-Leong Chew 7, Marko Lampe 8, Arnat Balabiyev 1,2, Robert Ros 3,4,5, Tatiana P. Ugarova 1,2
1Center for Metabolic and Vascular Biology, Mayo Clinic, 2Molecular and Cellular Biosciences, School of Life Sciences, Arizona State University, 3Department of Physics, Arizona State University, 4Center for Biological Physics, Arizona State University, 5Biodesign Institute, Arizona State University, 6Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 7Advanced Imaging Center, HHMI Janelia Research Campus, 8Advanced Light Microscopy Facility, European Molecular Biology Laboratory

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

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Medicine

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish
Xiao Ma *1,2,3, Yonghe Ding *2,3, Yong Wang 2,3,4, Xiaolei Xu 1,2,3
1Clinical and Translational Sciences Track, Mayo Clinic Graduate School of Biomedical Sciences, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, 3Division of Cardiovascular Diseases, Mayo Clinic, 4Institute of Life Science, Beijing University of Chinese Medicine

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

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Biology

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells
Katilyn Knutson *1, Peter R. Strege *1, Joyce Li 2, Andrew B. Leiter 2, Gianrico Farrugia 1, Arthur Beyder 1
1Enteric Neuroscience Program, Division of Gastroenterology & Hepatology,Department of Physiology & Biomedical Engineering, Mayo Clinic, 2Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

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Medicine

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine
Robert A. Kaiser 1,2, Shennen A. Mao 1, Jaime Glorioso 3, Bruce Amiot 1, Clara T. Nicolas 1, Kari L. Allen 1, Zeji Du 1, Caitlin J VanLith 1, Raymond D. Hickey 1, Scott L. Nyberg 1, Joseph B. Lillegard 1,2,4
1Department of Surgery, Mayo Clinic, 2Midwest Fetal Care Center, Children's Hospitals and Clinics of Minnesota, 3Department of Surgery, Johns Hopkins, 4Pediatric Surgical Associates

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

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JoVE Core

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans
Courtney M. Wheatley 1, Triven Kannan 1, Svetlana Bornschlegl 2, Chul-ho Kim 1, Dennis A. Gastineau 2, Allan B. Dietz 2, Bruce D. Johnson 1, Michael P. Gustafson 2
1Department of Cardiovascular Diseases, Mayo Clinic, 2Department of Laboratory Medicine and Pathology, Mayo Clinic

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

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Biochemistry

Isolation of Tissue Extracellular Vesicles from the Liver
Kaori Ishiguro 1, Irene K. Yan 1, Tushar Patel 1
1Department of Transplantation, Mayo Clinic

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

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Effective Lysis of Cyanobacterial and Green Algal Single Cells for Whole Genome Amplification in Microfluidics
Yuguang Liu 1, Dirk Schulze-Makuch 2, Jean-Pierre de Vera 3, Charles Cockell 4, Thomas Leya 5, Mickael Baque 3, Marina Walther-Antonio 1,6
1Department of Surgery, Division of Surgical Research, Mayo Clinic, 2Astrobiology Group, Center of Astronomy and Astrophysics, Technical University, 3Management and Infrastructure, Astrobiological Laboratories, German Aerospace Center (DLR), Institute of Planetary Research, 4School of Physics and Astronomy, University of Edinburgh, 5Branch Bioanalytics and Bioprocesses (IZI-BB), Extremophile Research & Biobank CCCryo, Fraunhofer Institute for Cell Therapy and Immunology, 6Department of Obstetrics and Gynecology, Mayo Clinic

Here we present a protocol to lyse cyanobacteria and green algae single cells that allows for subsequent single-cell whole genome amplification in a microfluidic platform with a 100% success rate.

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Using Structural DNA Alteration Analysis in Conjunction with Patient-Derived Xenografts to Test Targeted Cancer Therapies
Lin Yang 1, Faye R. Harris 1, Piyan Zhang 1, George Vasmatzis 1,2, Irina V. Kovtun 1,3
1Center for Individualized Medicine, Mayo Clinic, 2Molecular Medicine, Mayo Clinic, 3Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic

Presented here is a protocol to test the efficacy of targeted therapies that are selected based on the genomic makeup of tumors. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patient tumors into mice, and testing of responses to corresponding drugs.

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Cancer Research

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells
Rosalie M. Sterner 1,2, Michelle J. Cox 3, Reona Sakemura 3, Saad S. Kenderian 2,3
1Mayo Clinic Medical Scientist Training Program, Mayo Clinic College of Medicine and Science, 2Department of Immunology, Mayo Clinic, 3Division of Hematology, Mayo Clinic

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

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Developmental Biology

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development
Yong Wang *1, Ding Zhang *2, Fang Zhou 2, Meijun Zhou 2, Qiujie Li 1, Jingyu Chen 3, Jun Yang 1,2
1Department of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, 2Department of Cell Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Wuxi Lung Transplant Center, Wuxi People's Hospital affiliated to Nanjing Medical University

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

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JoVE Core

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts
Piyan Zhang 1, Irina V. Kovtun 1,2
1Center for Individualized Medicine, Mayo Clinic, 2Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients’ tumors into mice and testing responses to corresponding drugs.

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Medicine

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube
Dmitry Frank *1, Shiri Savir *1, Benjamin F. Gruenbaum 2, Israel Melamed 3, Julia Grinshpun 1, Ruslan Kuts 1, Boris Knyazer 4, Alexander Zlotnik 1, Max Vinokur 1, Matthew Boyko 1
1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology, Yale University School of Medicine, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

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Genetics

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)
Yaolei Mi 1,2, Zhihui Zhu 3, Guangtao Qian 3, Yu Li 4, Xiangxiao Meng 1, Jianping Xue 3, Qingfu Chen 5, Wei Sun 1, Yuhua Shi 1,2
1Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, 2Artemisinin Research Center, China Academy of Chinese Medical Sciences, 3College of Life Science, Huaibei Normal University, 4Economic Crop Research Institute Sichuan Academy of Agriculture Sciences, 5Research Center of Buckwheat Industry Technology, Guizhou Normal University

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

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Neuroscience

Laser-Induced Brain Injury in the Motor Cortex of Rats
Ruslan Kuts *1, Israel Melamed *2, Honore N. Shiyntum 3, Benjamin F. Gruenbaum 4, Dmitry Frank 1, Boris Knyazer 5, Dmitry Natanel 1, Olena Severynovska 3, Max Vinokur 1, Matthew Boyko 1
1Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Biochemistry and Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro National University, 4Department of Anesthesiology, Yale University School of Medicine, 5Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

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Neuroscience

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration
Dmitry Frank *1, Israel Melamed *2, Benjamin F. Gruenbaum 3, Julia Grinshpun 1, Ruslan Kuts 1, Rachel Shvartsur 4, Abed N. Azab 4, Mohamad H. Assadi 5, Max Vinokur 1, Matthew Boyko 1
1Division of Anesthesia and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Anesthesiology, Yale University School of Medicine, 4Recanati School for Community Health Professions, Faculty of Health Sciences, Ben-Gurion University of the Negev, 5Department of microbiology and immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

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Neuroscience

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples
Dmitry Frank *1, Benjamin F. Gruenbaum *2, Julia Grinshpun 1, Israel Melamed 3, Olena Severynovska 4, Ruslan Kuts 1, Michael Semyonov 1, Evgeni Brotfain 1, Alexander Zlotnik 1, Matthew Boyko 1
1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology, Yale University School of Medicine, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Physiology, Faculty of Biology, Ecology and Medicine, Dnepropetrovsk State University

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

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Medicine

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model
Michaela Olthoff 1, Federico Franchi 1, Karen M. Peterson 1, Ramasamy Paulmurugan 2, Martin Rodriguez-Porcel 1
1Department of Cardiovascular Medicine, Mayo Clinic, 2Department of Radiology and Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

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Biology

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues
Dong Seong Cho 1, Jason D. Doles 1
1Department of Biochemistry and Molecular Biology, Mayo Clinic

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

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Behavior

A Complex Diving-For-Food Task to Investigate Social Organization and Interactions in Rats
Benjamin F. Gruenbaum *1, Dmitry Frank *2, Shiri Savir 2, Honore N. Shiyntum 3, Ruslan Kuts 2, Max Vinokur 2, Israel Melamed 4, Michael Dubilet 2, Alexander Zlotnik 2, Matthew Boyko 2
1Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 2Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Biophysics and Biochemistry, Oles Honchar Dnipro National University, 4Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

This protocol describes a method of examining social hierarchy in a rat model. Rats perform a complex diving-for-food task in which they form a distinct hierarchy according to their willingness to dive underwater and swim to obtain a food pellet. This method is used to understand decision making and social relationships among highly social animals in small groups.

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JoVE Core

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy
Nicholas R. Jaegers 1, Wenda Hu 2, Yong Wang 1, Jian Zhi Hu 1
1Pacific Northwest National Laboratory, 2Washington State University

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

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Neuroscience

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat
Meiwand Bedar 1,2, Tiam M. Saffari 1,2, Patricia F. Friedrich 1, Guilherme Giusti 1, Allen T. Bishop 1, Alexander Y. Shin 1
1Department of Orthopedic Surgery, Mayo Clinic, 2Radboud University Medical Center, Radboud Institute for Health Sciences, Department of Plastic Surgery, Nijmegen, The Netherlands

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

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Medicine

Veno-Arterial Extracorporeal Membrane Oxygenation for Cardiogenic Shock
Aravdeep Jhand 1, Muhammad Asim Shabbir 2, John Um 3, Poonam Velagapudi 2
1Cardiovascular Medicine, Mayo Clinic, 2Cardiovascular Medicine, University of Nebraska Medical Center, 3Cardiothoracic Surgery, University of Nebraska Medical Center

The following article highlights various steps involved in initiating and maintaining veno-arterial extracorporeal membrane oxygenation in patients with cardiogenic shock.

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Neuroscience

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury
Dmitry Frank *1, Benjamin F. Gruenbaum *2, Israel Melamed 3, Julia Grinshpun 1, Yair Benjamin 1, Ievgeni Vzhetson 1, Nadia Kravchenko 4, Michael Dubilet 1, Matthew Boyko *1, Alexander Zlotnik *1
1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Physiology, Faculty of Biology, Ecology and Medicine, Dnepropetrovsk State University

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

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Cancer Research

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography
Reona Sakemura 1,2, Michelle J. Cox 1,2, Aditya Bansal 3, Claudia Manriquez Roman 1,2,4,5, Mehrdad Hefazi 1,2, Cynthia J. Vernon 3, Dianna L. Glynn 3, Mukesh K. Pandey 3, Timothy R. DeGrado 3, Elizabeth L. Siegler 1,2, Saad S. Kenderian 1,2,5,6
1T Cell Engineering, Mayo Clinic, 2Division of Hematology, Mayo Clinic, 3Department of Radiology, Mayo Clinic, 4Regenerative Sciences PhD, Mayo Clinic, 5Department of Molecular Medicine, Mayo Clinic, 6Department of Immunology, Mayo Clinic

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

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Cancer Research

Zebrafish Model of Neuroblastoma Metastasis
Zuag Paj Her 1, Kok Siong Yeo 1, Cassie Howe 1, Taylor Levee 1, Shizhen Zhu 1
1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic Cancer Center

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

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Medicine

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys
Heather L. Holmes *1, Alison A. Stiller *1, Christopher J. Moore 4, Adriana V. Gregory 2, Carolyn M. Roos 3, Jordan D. Miller 1,3, Ryan C. Gessner 4, Tomasz J. Czernuszewicz 4, Timothy L. Kline 2, Michael F. Romero 1,5
1Physiology & Biomedical Engineering, Mayo Clinic, 2Radiology, Mayo Clinic, 3Surgery, Mayo Clinic, 4SonoVol, Inc., 5Nephrology & Hypertension, Mayo Clinic

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

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Bioengineering

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids
Kihak Gwon *1, Hye Jin Hong *1, Alan M. Gonzalez-Suarez 1, Gulnaz Stybayeva 1, Alexander Revzin 1
1Department of Physiology and Biomedical Engineering, Mayo Clinic

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

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Biology

Enhancing Tumor Content through Tumor Macrodissection
Lee Wisner 1, Brandon Larsen 2, Alanna Maguire 1
1Department of Research, Mayo Clinic, 2Department of Laboratory Medicine and Pathology, Mayo Clinic

This protocol presents a method to increase the percent tumor content of formalin-fixed paraffin-embedded tissue samples.

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Biology

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells
Qianqian Guo 1, Kunimaro Furuta 1, Ahmed Aly 1, Samar H. Ibrahim 1,2
1Division of Gastroenterology & Hepatology, Mayo Clinic, 2Division of Pediatric Gastroenterology, Mayo Clinic

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

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Cancer Research

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer
Lee Wisner 1, Brandon Larsen 2, Alanna Maguire 1
1Department of Research, Mayo Clinic, 2Department of Laboratory Medicine and Pathology, Mayo Clinic

This protocol outlines the tape method on how to manually construct a tissue microarray using FFPE donor blocks of differing depths.

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Bioengineering

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood
Akankshya Shradhanjali 1, Susheil Uthamaraj 2, Dan Dragomir-Daescu 3, Rajiv Gulati 4, Gurpreet S. Sandhu 4, Brandon J. Tefft 1
1Department of Biomedical Engineering, Medical College of Wisconsin & Marquette University, 2Division of Engineering, Mayo Clinic, 3Department of Physiology & Biomedical Engineering, Mayo Clinic, 4Department of Cardiovascular Medicine, Mayo Clinic

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

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Unfixed Head and Closed Skull Weight Drop Method as a Rodent Model of Mild Traumatic Brain Injury (Mtbi) for Studies of Post-Traumatic Headache (PTH)
Caroline Machado Kopruszinski 1, Trent Anderson 2, Janice Oyarzo 3, Edita Navratilova 1,3, Frank Porreca 1,3
1Department of Pharmacology, College of Medicine, University of Arizona, Tucson, 2Department of Basic Medical Sciences, College of Medicine, University of Arizona, Phoenix, 3Department of Collaborative Research, Mayo Clinic

The protocol describes a rodent model of unfixed head mild traumatic brain injury induced by a weight drop allowing for free linear and rotational impact-related forces. The dynamics of this injury are similar to concussions that occur in humans and are useful to investigate underlying mechanisms involved in post-traumatic headache.

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Medicine

Studying Murine Small Bowel Mechanosensing of Luminal Particulates
Arnaldo Mercado-Perez *1,2, Andrew Wegner *1, Kaitlyn Knutson 1, Michael Zumchak 3, Arthur Beyder 1,4
1Enteric NeuroScience Program (ENSP), Division of Gastroenterology & Hepatology, Department of Medicine, Mayo Clinic, 2Medical Scientist Training Program (MSTP), Mayo Clinic, 3Easy Learning Labs, 4Department of Physiology & Biomedical Engineering (PBME), Mayo Clinic

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

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Behavior

A Protocol for the Induction of Posttraumatic Stress-Disorder (PTSD)-like Behavior in Mice
Graeme Preston 1, Tamas Kozicz 1
1Department of Clinical Genomics, Mayo Clinic

In this study, post-traumatic stress disorder (PTSD)-like behavior is induced in mice using two sessions of inescapable electric foot shock. PTSD-like and resilient animals are identified using several assays for PTSD-specific behaviors.

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Cancer Research

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling
Aditya Bansal 1, Timothy R. DeGrado 2, Mukesh K. Pandey 1
1Division of Nuclear Medicine, Department of Radiology, Mayo Clinic, 2Department of Radiology, University of Colorado Anschutz Medical Campus

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

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Behavior

Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury
Dmitry Frank *1, Benjamin F. Gruenbaum *2, Michael Semyonov 1, Yair Binyamin 1, Olena Severynovska 3, Ron Gal 1, Amit Frenkel 1, Boris Knazer 4, Matthew Boyko 1, Alexander Zlotnik 1
1Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 3Department of Biochemistry and Physiology of the Faculty of Biology and Ecology, Oles Gonchar of the Dnipro National University, 4Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.

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Medicine

Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis
Rebecca G. Theophanous 1, Vinca W. Chow 2, David L. Convissar 3, Stephen C. Haskins 4,5, Robert A. Jones 6, Hari K. P. Kalagara 7, Yuriy S. Bronshteyn 8
1Department of Emergency Medicine, Duke University School of Medicine, 2Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, 3Department of Anesthesiology, Massachusetts General Hospital, 4Department of Anesthesiology, Critical Care and Pain Management, Hospital for Special Surgery, 5Department of Anesthesiology, Weill-Cornell Medical College, 6Department of Emergency Medicine, MetroHealth Medical Center/Case Western Reserve University, 7Department of Anesthesiology, Mayo Clinic, 8Department of Anesthesiology, Duke University School of Medicine

Traditionally, lower extremity deep venous thrombosis (DVT) is diagnosed by radiology-performed venous duplex ultrasound. Providers appropriately trained in focused point-of-care ultrasound (POCUS) can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. We describe the scanning technique for focused POCUS DVT lower extremity examination.

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Medicine

A Simple Cuff Technique for Murine Left Lung Transplantation
Zhongcheng Mei 1, Mojtaba Taheri 1, Elizabeth A. Jacobsen 2, Wenjun Li 3,4, Andrew E. Gelman 3,4, Daniel Kreisel 3,4, Alexander Sasha Krupnick 1,5
1Department of Surgery, University of Maryland Medical Center, 2Division of Allergy, Asthma and Clinical Immunology, Mayo Clinic, 3Department of Surgery, Washington University in St. Louis, 4Department of Pathology & Immunology, Washington University in St. Louis, 5Department of Microbiology and Immunology, University of Maryland Medical Center

The present protocol describes a cuff technique for a mouse left lung transplantation model. This technique has been developed over several years and has performed well, serving effectively in immunological research.

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Medicine

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality
Maryam Moossavi *1,2, Hong Zhang *3,4, Jiarong Li 1,2,4, Feixiang Yan 1,2, Xiaolei Xu 1,2
1Department of Biochemistry and Molecular Biology, Mayo Clinic, 2Department of Cardiovascular Medicine, Mayo Clinic, 3Cardiovascular Surgery Department, Guilin Hospital of 2nd Xiangya Hospital, Central South University, 4Cardiovascular Surgery Department, 2nd Xiangya Hospital, Central South University

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

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Neuroscience

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats
Obaid U. Khurram 1, Matthew J. Fogarty 1, Wen-Zhi Zhan 1, Carlos B. Mantilla 1,2, Gary C. Sieck 1,2
1Department of Physiology and Biomedical Engineering, Mayo Clinic, 2Department of Anesthesiology and Perioperative Medicine, Mayo Clinic

Respiratory complications are the leading cause of death in individuals with cervical spinal cord injury (cSCI). Animal models of cSCI are essential for mechanistic evaluations and pre-clinical studies. Here, we introduce a reproducible method to assess functional recovery of diaphragm muscle (DIAm) activity following unilateral C2 spinal hemisection (C2SH) in rats.

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Bioengineering

Zebra II as A Novel System to Record Electrophysiological Signals in Zebrafish
Ramses Seferino Trigo Torres 1, Mao-Hsiang Huang 2, Mohamed Benomar 2, Tai Le 1, Tim Etchells 3, Xiaolei Xu 4, Michael P. H. Lau 3, Hung Cao 1,2
1Department of Biomedical Engineering, UC Irvine, 2Department of Electrical Engineering and Computer Science, UC Irvine, 3Sensoriis Inc., 4Department of Biochemistry and Molecular Biology, Mayo Clinic

The study introduces Zebra II, an advanced system for prolonged ECG acquisition and analysis in zebrafish. This system features an independent perfusion system and multiple-point electrodes for handling multiple fish in controlled environments. Acute effects of Amiodarone were assessed and analyzed with wild-type zebrafish.

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Biochemistry

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay
Intisar Koch 1, Matthew J. Schellenberg 2, Francisco Bustos 1,3
1Pediatrics and Rare Diseases Group, Sanford Research, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, 3Department of Pediatrics, Sanford School of Medicine, University of South Dakota

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

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