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University of Colorado Denver

22 ARTICLES PUBLISHED IN JoVE

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Medicine

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice
Michael Koeppen 1, Tobias Eckle 1, Holger K. Eltzschig 1
1Department of Anesthesiology, University of Colorado

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

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Medicine

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice
Tobias Eckle 1, Michael Koeppen 1, Holger Eltzschig 1
1Department of Anesthesiology, University of Colorado Denver

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

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Medicine

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury
Michael P. Hutchens 1, Richard J. Traystman 2, Tetsuhiro Fujiyoshi 1, Shin Nakayama 1, Paco S. Herson 1
1Department of Anesthesiology and Perioperative Medicine, Oregon Health & Sciences University, 2Department of Pharmacology, University of Colorado Denver

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

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Biology

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells
Kelsey S. Chow 1,2, DuHyun Jun 1,2, Karen M. Helm 3, David H. Wagner 1,2,4, Susan M. Majka 1,2,3
1Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, 2Department of Medicine, University of Colorado Denver, 3Cancer Center, University of Colorado Denver, 4Webb Waring Institute, University of Colorado Denver

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

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Medicine

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer
Yimu Yang 1, Gaoqing Yang 1, Eric P. Schmidt 1
1Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

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Neuroscience

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Stacy L. Fairbanks *1, Rebekah Vest *1, Saurabh Verma 2, Richard J. Traystman 1,3, Paco S. Herson 1,3
1Department of Anesthesiology, University of Colorado School of Medicine, 2Oregon National Primate Research Center, Oregon Health & Science University, 3Department of Pharmacology, University of Colorado School of Medicine

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

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Medicine

Murine Heterotopic Heart Transplant Technique
Robert J. Plenter 1, Todd J. Grazia 1
1Colorado Center for Transplantation Care, Research and Education and Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

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Medicine

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats
Gert Blueschke *1, Gabi Hanna *2, Andrew N. Fontanella 2, Gregory M. Palmer 2, Alina Boico 2, Hooney Min 2, Mark W. Dewhirst 2, David C. Irwin 3, Yulin Zhao 2, Thies Schroeder 4
1Division of Plastic, Maxillofacial, and Oral Surgery, Duke University Medical Center, 2Department of Radiation Oncology, Duke University Medical Center, 3Department of Cardiology, University of Colorado Denver, 4Department of Physical Chemistry, University of Mainz

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

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Bioengineering

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Charmion Cruickshank-Quinn 1, Kevin D. Quinn 1, Roger Powell 1, Yanhui Yang 1, Michael Armstrong 1, Spencer Mahaffey 2, Richard Reisdorph 1, Nichole Reisdorph 1
1Department of Immunology, National Jewish Health, 2Department of Pharmacology, School of Medicine, University of Colorado Denver

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

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Neuroscience

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae
Tiffany M. Nuessle 1, Nicole L. Garneau 1, Meghan M. Sloan 1, Stephanie A. Santorico 2
1Health Sciences Department, Denver Museum of Nature & Science, 2Department of Mathematical and Statistical Sciences, University of Colorado Denver

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller & Reedy (1990).

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Developmental Biology

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells
Michael Ludwig 1, David Kitzenberg 1, Wallace S. Chick 1
1Department of Cell and Developmental Biology, University of Colorado Denver

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

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Chemistry

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction
Mohand O. Saed 1, Amir H. Torbati 1,2, Devatha P. Nair 2, Christopher M. Yakacki 1
1Department of Mechanical Engineering, University of Colorado Denver, 2Department of Ophthalmology, University of Colorado Denver

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

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Chemistry

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism
Andrew J. Francis 1, Marino J. E. Resendiz 1
1Department of Chemistry, University of Colorado Denver

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2'-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

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Medicine

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse
Marilee J. Wick 1,2, Julie W. Harral 1,2, Zoe L. Loomis 1,2, Edward C. Dempsey 1,2,3
1Cardiovascular Pulmonary Research Laboratory, University of Colorado Denver, 2Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, 3Denver VA Medical Center

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

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Immunology and Infection

Electrochemiluminescence Assays for Human Islet Autoantibodies
Yong Gu 1,2, Zhiyuan Zhao 1, Dongmei Miao 1, Hilary High 1, Tao Yang 2, Liping Yu 1
1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 2Department of Endocrinology, First Affiliated Hospital of Nanjing Medical University

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

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Neuroscience

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks
Katelyn B. Sondereker 1, Maureen E. Stabio 2, Jenna R. Jamil 1, Matthew J. Tarchick 1, Jordan M. Renna 1
1Department of Biology, The University of Akron, 2Department of Cell and Developmental Biology, University of Colorado Denver

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

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Medicine

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung
Sumei Liao 1, Oliver Eickelberg 1, Eric P. Schmidt 1,2, Yimu Yang 1
1Department of Medicine, University of Colorado Denver, 2Department of Medicine, Denver Health Medical Center

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.  In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.  This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

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Immunology and Infection

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model
Howard W. Davidson 1, Joseph Ray Cepeda 2, Nitin S. Sekhar 2, Junying Han 2, Ling Gao 3, Tomasz Sosinowski 1, Li Zhang 2
1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 2Department of Medicine, Endocrinology, Diabetes & Metabolism, Baylor College of Medicine, 3Scientific Center, Shandong Provincial Hospital affiliated to Shandong University

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

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Medicine

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases
Xiaofan Jia *1,2, Ling He *1,3, Yong Gu 1, Hilary High 1, Liping Yu 1
1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, 2Department of Endocrinology, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, 3Department of endocrinology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

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Biology

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining
Wells B. LaRiviere 1,2, Xiaorui Han 3,4, Kaori Oshima 1, Sarah A. McMurtry 1, Robert J. Linhardt 3, Eric P. Schmidt 1,5
1Department of Medicine, University of Colorado Anschutz Medical Campus, 2Medical Scientist Training Program, University of Colorado Anschutz Medical Campus, 3Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 4Department of Health Sciences, Curtin University, 5Department of Medicine, Denver Health Medical Center

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

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Immunology and Infection

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19
Xiaofan Jia *1, Ling He *1,2, Dongmei Miao 1, Caiguo Zhang 1, Marian Rewers 1, Liping Yu 1
1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, 2Department of Endocrinology, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

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Biology

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria
Rihab Bouchareb 1, Liping Yu 1, Emelie Lassen 1, Ilse S. Daehn 1
1Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

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