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The Ohio State University College of Medicine

17 ARTICLES PUBLISHED IN JoVE

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Medicine

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function
Li Zuo 1,2, William J. Roberts 1,2, Kevin D. Evans 1
1Radiologic Sciences & Respiratory Therapy Division, School of Health and Rehabilitation Sciences, Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, 2Department of Biological Sciences, Oakland University

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

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Medicine

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program
Kevin Nelson 1,2, Christopher Bobba 1,2, Emre Eren 3, Tyler Spata 4, Malak Tadres 2, Don Hayes, Jr. 5,6, Sylvester M. Black 3,7, Samir Ghadiali *1,2,3, Bryan A. Whitson *2,3,4
1Department of Biomedical Engineering, Ohio State University Wexner Medical Center, 2Davis Heart & Lung Research Institute, Ohio State University Wexner Medical Center, 3The Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Laboratory, Ohio State University Wexner Medical Center, 4Division of Cardiac Surgery, Department of Surgery, Ohio State University Wexner Medical Center, 5Departments of Pediatrics and Internal Medicine, Ohio State University, 6Advanced Lung Disease Program, Lung and Heart-Lung Transplant Programs, Nationwide Children's Hospital, 7Division of Transplantation, Department of Surgery, Ohio State University Wexner Medical Center

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

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Behavior

Mindfulness in Motion (MIM): An Onsite Mindfulness Based Intervention (MBI) for Chronically High Stress Work Environments to Increase Resiliency and Work Engagement
Maryanna Klatt 1, Beth Steinberg 2, Anne-Marie Duchemin 3
1Department of Family Medicine, The Ohio State University College of Medicine, 2Critical Care Nursing, Wexner Medical Center, 3Department of Psychiatry, Stress, Trauma, and Resilience (STAR) Program, The Ohio State University College of Medicine

The Mindfulness in Motion (MIM) protocol offers a pragmatic Mindfulness Based Intervention (MBI) on-site, for persons working in chronically high-stress work environments that significantly increases resiliency and work engagement. The protocol has proven feasible, beneficial, and is easily adaptable to other high-stress workplaces.

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Biology

Isolation of Murine Coronary Vascular Smooth Muscle Cells
Kathryn E. Husarek 1,2, Xiaojin Zhang 2, Patricia E. McCallinhart 2, Pamela A. Lucchesi 2,3, Aaron J. Trask 2,3
1School of Biomedical Science, The Ohio State University College of Medicine, 2Center for Cardiovascular and Pulmonary Research, The Research Institute at Nationwide Children's Hospital, 3Department of Pediatrics, The Ohio State University College of Medicine

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

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Developmental Biology

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae
Elliott J. Hagedorn 1,2, Jennifer L. Cillis 3, Caitlyn R. Curley 3, Taylor C. Patch 3, Brian Li 1,2, Bradley W. Blaser 1,2,7, Raquel Riquelme 1,2, Leonard I. Zon 1,2,4,5,6, Dhvanit I. Shah 1,2,3,4,5
1Division of Hematology/Oncology, Boston Children’s Hospital, 2Harvard Medical School, 3Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

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Medicine

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model
Eliza W. Beal 1, Curtis Dumond 1, Jung-Lye Kim 1, Khalid Mumtaz 1, Don Hayes Jr. 1, Ken Washburn 1, Bryan A. Whitson 1, Sylvester M. Black 1
1Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Lab, Division of Transplant, Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

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Immunology and Infection

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase
Miguel A. Lopez Jr. 1, Randi M. Mackler 1, Matthew P. Altman 1, Kristine E. Yoder 1
1Department of Cancer Biology and Genetics, The Ohio State University College of Medicine

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

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Immunology and Infection

Assembly and Purification of Prototype Foamy Virus Intasomes
Randi M. Mackler 1, Miguel A. Lopez Jr. 1, Kristine E. Yoder 1
1Department of Cancer Biology and Genetics, The Ohio State University College of Medicine

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

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Medicine

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion
Eliza W. Beal 1,2, Curtis Dumond 1, Jung-Lye Kim 1,2, Clifford Akateh 1,2, Emre Eren 1, Katelyn Maynard 1, Chandan K. Sen 3, Jay L. Zweier 4, Kenneth Washburn 2, Bryan A. Whitson 1,3, Sylvester M. Black 1,2
1Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Lab, Division of Transplant, Department of Surgery, Comprehensive Transplant Center, Ohio State University Wexner Medical Center, 2Department of Surgery, Division of Transplant, Ohio State University Wexner Medical Center, 3Department of Surgery, Division of CardioThoracic Surgery, Ohio State University Wexner Medical Center, 4Department of Medicine, Ohio State University Wexner Medical Center

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

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Bioengineering

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model
Matthew G. Wiet *1,2, Sayali Dharmadhikari *1,3, Audrey White 1,2, Susan D. Reynolds 4, Jed Johnson 5, Christopher K. Breuer 3,6, Tendy Chiang 1,3
1Department of Otolaryngology Head & Neck Surgery, Nationwide Children's Hospital, 2The Ohio State University College of Medicine, 3Center for Regenerative Medicine, Research Institute at Nationwide Children's Hospital, 4Center for Perinatal Research, Nationwide Children's Hospital, 5Nanofiber Solutions, Inc., 6Department of Pediatric Surgery, Nationwide Children's Hospital

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

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Biochemistry

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase
Ryan K. Messer 1, Miguel A. Lopez Jr. 1, Gayan Senavirathne 1, Kristine E. Yoder 1
1Cancer Biology and Genetics, College of Medicine, The Ohio State University

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

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JoVE Journal

Analysis of Hematopoietic Stem Progenitor Cell Metabolism
Giorgia Scapin 1,2,3, Marie C. Goulard 1,2,3, Priyanka R. Dharampuriya 1,2,3, Jennifer L. Cillis 1,2,3, Dhvanit I. Shah 1,2,3
1Nationwide Children's Hospital, 2The Ohio State University College of Medicine, 3The Ohio State University Comprehensive Cancer Center

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

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Developmental Biology

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells
Shiqiao Ye 1,2, Xiaoping Wan 3, Juan Su 1,2, Akshar Patel 1,2,4, Blake Justis 1,2,5, Isabelle Deschênes 3, Ming-Tao Zhao 1,2,5,6
1Center for Cardiovascular Research, The Abigail Wexner Research Institute, Nationwide Children’s Hospital, 2The Heart Center, Nationwide Children’s Hospital, 3Department of Physiology and Cell Biology, The Ohio State University College of Medicine, 4Department of Anatomy, The Ohio State University College of Medicine, 5MCDB Graduate Program, The Ohio State University, 6Department of Pediatrics, The Ohio State University College of Medicine

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

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Medicine

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes
Yong Gyu Lee 1,2, Jung-Lye Kim 1,2, Andre F. Palmer 3, Brenda F. Reader 4, Jianjie Ma 1, Sylvester M. Black 2,4, Bryan A. Whitson 1,2
1Department of Surgery Division of Cardiac Surgery, The Ohio State University Wexner Medical Center, 2The Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Laboratory, The Ohio State University College of Medicine, 3William G. Lowrie Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, 4Comprehensive Transplant Center, The Ohio State University Wexner Medical Center

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

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Biochemistry

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay
Pavan S. Krishnan 1,2, Fernando T. Zamuner 1,3, Carolyn M. Jenks 1, Johnny Y. Xie 4, Lisa Zhang 5, Mohammed Lehar 1, Neal S. Fedarko 6, Mariana Brait 1,3, John P. Carey 1
1Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine, 2Virginia Commonwealth University School of Medicine, 3Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 4Department of Otolaryngology-Head and Neck Surgery, University of Michigan Medical School, 5Department of Otolaryngology-Head and Neck Surgery, The Ohio State University College of Medicine, 6ICTR Clinical Research Core Laboratory, Johns Hopkins University School of Medicine

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

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Medicine

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography
Qiang Zeng 1, Doug A. Gouchoe 2,3, Mahboubeh Nabavinia 4, Yong Gyu Lee 2, Xi Wang 2, Terri A. Shaffer 5, Mitchel R. Stacy 4,5,6, Blake R. Peterson 7,8, Bryan A. Whitson 2,9, Christopher Breuer 1,4, Sylvester M. Black 1,10
1Department of Surgery, Nationwide Children’s Hospital, 2COPPER Laboratory, The Ohio State University, 388th Surgical Operation Operations Squadron, WPAFB, 4Center for Regenerative Medicine, The Research Institute at Nationwide Children’s Hospital, 5Division of Vascular Diseases and Surgery, The Ohio State University College of Medicine, 6Interdisciplinary Biophysics Graduate Program, The Ohio State University, 7Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University College of Pharmacy, 8The Ohio State University Comprehensive Cancer Center, 9Division of Cardiac Surgery, Department of Surgery, The Ohio State Wexner Medical Center, 10Division of Transplantation, Department of Surgery, The Ohio State Wexner Medical Center

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

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Bioengineering

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography
Delaney J. Villarreal *1,2, Tatsuya Watanabe *1, Kirsten Nelson 1, Adrienne Morrison 1, Eric D. Heuer 1, Anudari Ulziibayar 1, John M. Kelly 1,3,4, Christopher K. Breuer 1,5
1Center for Regenerative Medicine, Nationwide Children's Hospital, 2Biomedical Sciences Graduate Program, The Ohio State University College of Medicine, 3The Heart Center, Nationwide Children's Hospital, 4Department of Pediatrics, The Ohio State University College of Medicine, 5Department of Surgery, The Ohio State University College of Medicine

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

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