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Oregon Health & Science University

27 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5
Maria Pia Testa 1, Omar Alvarado 1, Andrea Wournell 1, Jonathan Lee 2, Frederick T. Guilford 3, Steven H. Henriksen 2, Tom R. Phillips 1,2
1College of Veterinary Medicine, Western University of Health Sciences, 2Graduate College of Biomedical Sciences, Western University of Health Sciences, 3ReadiSorb, Products

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

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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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Neuroscience

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation
Lingyan Wang 1, Han Jiang 1, John V. Brigande 1
1Oregon Hearing Research Center, Oregon Health & Science University

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

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Medicine

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring
Yingding Xu 1, Hongguang Liu 1, Edwin Chang 1, Han Jiang 1, Zhen Cheng 1
1Department of Radiology and Bio-X Program Canary Cancer at Stanford for Cancer Early Detection, Stanford University

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

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Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization
Leslie Smith 1, Mathew Thayer 1
1Department of Biochemistry and Molecular Biology, Knight Cancer Institute, Oregon Health & Science University

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

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Biology

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
Katharina L. Dürr 1,2, Neslihan N. Tavraz 1, Susan Spiller 1, Thomas Friedrich 1
1Institute of Chemistry, Technical University of Berlin, 2The Vollum Institute, Oregon Health & Science University

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

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Immunology and Infection

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Samuel L. Drennan 1, Amrita Lama 1, Ben Doron 1, Eric D. Cambronne 1
1Department of Molecular Microbiology & Immunology, Oregon Health & Science University

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

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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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Neuroscience

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons
Robert P. Irwin 1, Charles N. Allen 1,2
1Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, 2Department of Behavioral Neuroscience, Oregon Health & Science University

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

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Biology

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle
William C.W. Chen 1, Arman Saparov 2,3, Mirko Corselli 4, Mihaela Crisan 5, Bo Zheng 6, Bruno Péault 7,8, Johnny Huard 9
1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine, Queen's Medical Research Institute and University of Edinburgh, 8David Geffen School of Medicine and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 9Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

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Bioengineering

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Esra Güç *1, Manuel Fankhauser *1, Amanda W. Lund 1,2, Melody A. Swartz 1, Witold W. Kilarski 1
1Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, 2Department of Cell and Developmental Biology and Knight Cancer Institute, Oregon Health & Science University

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

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Neuroscience

Super-resolution Imaging of Neuronal Dense-core Vesicles
Bethe A. Scalettar 1,2, Daniel Shaver 2, Stefanie Kaech 3, Janis E. Lochner 2,4
1Department of Physics, Lewis & Clark College, 2Program in Biochemistry and Molecular Biology, Lewis & Clark College, 3Jungers Center for Neuroscience Research, Oregon Health & Science University, 4Department of Chemistry, Lewis & Clark College

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

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Biology

Visualizing the Early Stages of Phagocytosis
Ali Rashidfarrokhi *2, Veronica Richina *2, Fikadu G. Tafesse 1,2
1Department of Molecular Microbiology & Immunology, Oregon Health & Science University, 2Ragon Institute of MGH, MIT and Harvard

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

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Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram
Jonathan A. Coleman 1, Evan M. Green 2, Eric Gouaux 1,3
1Vollum Institute, Oregon Health & Science University, 2Graduate Group in Biophysics, University of California, San Francisco, 3Howard Hughes Medical Institute, Oregon Health & Science University

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

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Cancer Research

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis
Charles E. Gast 1, Aubie K. Shaw 1,2, Melissa H. Wong 1,3, Lisa M. Coussens 1,3
1Cell, Developmental & Cancer Biology, Oregon Health & Science University, 2University of Minnesota, 3Knight Cancer Institute, Oregon Health & Science University

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

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Medicine

Establishing a Multidisciplinary Cavernous Carotid Injury Simulation to Train Neurosurgical, Otolaryngology, and Anesthesia Residents
Brandon Lucke-Wold 1, Haley E. Gillham 2, Mark Baskerville 2, William E. Cameron 2, Dawn Dillman 2, Caleb A. Haley 2, Michele Noles 2, Donn Spight 2, Jeremy N. Ciporen 2
1West Virginia University School of Medicine, 2Oregon Health & Science University

This protocol establishes a multidisciplinary model to train learners on management of cavernous carotid artery injury. Cadaveric heads undergo expanded endonasal approach and injury to the cavernous carotid artery, and perfusion pump simulates blood flow to the injury point. Learners are tasked with medical and surgical management over 3 scenarios.

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Biology

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins
John M. Powers 1, Xiao Lan Chang 1, Zhen Song 1, Hiroyuki Nakai 1,2
1Department of Molecular and Medical Genetics, Oregon Health & Science University, 2Department of Molecular Microbiology and Immunology, Oregon Health & Science University

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

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Medicine

Development of a Cadaveric Multiport Model of Posterior Circulation Aneurysm Clipping for Neurosurgery and Otolaryngology Residents
Haley E. Gillham 1, Brandon Lucke-Wold 2, Aclan Dogan 1, Justin Cetas 1, William E. Cameron 1, Jeremy N. Ciporen 1
1Oregon Health & Science University, 2West Virginia University School of Medicine

A model protocol to train neurosurgery and otolaryngology resident learners on endoscopic transclival clipping of posterior circulation aneurysms is described. Two endoscopic approaches to access the silicone-injected or perfused posterior circulation of cadaveric heads are established for training. Learners are tasked with clipping of posterior circulation based on clinical scenarios.

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Behavior

Methodology for Establishing a Community-Wide Life Laboratory for Capturing Unobtrusive and Continuous Remote Activity and Health Data
Jeffrey Kaye 1, Christina Reynolds 1, Molly Bowman 1, Nicole Sharma 1, Thomas Riley 1, Ona Golonka 1, Jonathan Lee 1, Charlie Quinn 1, Zachary Beattie 1, Johanna Austin 1, Adriana Seelye 1, Katherine Wild 1, Nora Mattek 1
1Department of Neurology, ORCATECH - Oregon Center for Aging & Technology, Oregon Health & Science University

Unobtrusive sensors and pervasive computing technology incorporated into the daily home life of older adults enables meaningful health and activity changes to be recorded continuously for months to years, providing ecologically valid, high frequency, multi-domain data for research or clinical use.

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Biology

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy
Katsuyuki Matsushita 1, Kirsti Golgotiu 1, Daniel J. Orton 2, Richard D. Smith 2, Karin D. Rodland 2, Paul D. Piehowski 2, Michael P. Hutchens 1,3
1Anesthesiology & Perioperative Medicine, Oregon Health & Science University, 2Environmental and Biological Services Division, Pacific Northwest National Laboratory, 3Operative Care Division, Portland Veterans Affairs Medical Center

We present use of 2-photon microscopy to place a micropipette within Bowman's urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

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Biology

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors
Maria M. Steele 1, Madeline J. Churchill 2, Alec P. Breazeale 1, Ryan S. Lane 1, Nicholas A. Nelson 1, Amanda W. Lund 1,2,3,4
1Department of Cell, Developmental, & Cancer Biology, Oregon Health and Science University, 2Department of Molecular Microbiology & Immunology, Oregon Health and Science University, 3Department of Dermatology, Oregon Health and Science University, 4Knight Cancer Institute, Oregon Health and Science University

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from naïve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

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Biochemistry

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers
Yi-Xin Huo 1,2, Bo Zheng 1, Ning Wang 1, Yunpeng Yang 1, Xinxin Liang 1, Xiaoyan Ma 1
1Key Laboratory of Molecular Medicine and Biotherapy, School of Life Sciences, Beijing Institute of Technology, 2UCLA Institute for Technology Advancement (Suzhou)

This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.

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Neuroscience

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
Bart C. Jongbloets *1, Lei Ma *1, Tianyi Mao 1, Haining Zhong 1
1Vollum Institute, Oregon Health & Science University

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKARα, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

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Medicine

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy
Zhiqiang Hou 1, Yunpei Zhang 1, Lingling Neng 1, Jinhui Zhang, Xiaorui Shi 1
1Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

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Cancer Research

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency
Amani Alshaikh 1,2, Dmytro Grygoryev 3, Dove Keith 4, Brett Sheppard 4,5, Rosalie C Sears 4,6, Jungsun Kim 3,6, Abdenour Soufi 1
1Centre for Regenerative Medicine, Institute of Regeneration and Repair, Institute of Stem Cell Research, The University of Edinburgh, 2King Abdulaziz City for Science and Technology Health Sector (KACST), 3Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, 4Brenden-Colson Canter for Pancreatic Care, Oregon Health & Science University, 5Department of Surgery, Oregon Health & Science University, 6Department of Molecular & Medical Genetics, Oregon Health & Science University

The present protocol describes the reprogramming of Pancreatic Ductal Adenocarcinoma (PDAC) and normal pancreatic ductal epithelial cells into induced pluripotent stem cells (iPSCs). We provide an optimized and detailed, step-by-step procedure, from preparing lentivirus to establishing stable iPSC lines.

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Neuroscience

BrainBeats as an Open-Source EEGLAB Plugin to Jointly Analyze EEG and Cardiovascular Signals
Cédric Cannard 1,2, Helané Wahbeh 2,3, Arnaud Delorme 1,2,4
1Centre de Recherche Cerveau et Cognition (CerCo), CNRS, Toulouse III University, 2Institute of Noetic Sciences (IONS), 3Department of Neurology, Oregon Health & Science University, 4Swartz Center of Computational Neuroscience (SCCN), INC, UCSD

The BrainBeats toolbox is an open-source EEGLAB plugin designed to jointly analyze EEG and cardiovascular (ECG/PPG) signals. It includes heartbeat-evoked potentials (HEP) assessment, feature-based analysis, and heart artifact extraction from EEG signals. The protocol will aid in studying brain-heart interplay through two lenses (HEP and features), enhancing reproducibility and accessibility.

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Bioengineering

Production of Nanofibrillar Patterned Collagen for Tissue Engineering
Krista M. Habing *1, Yong How Tan *1, Karina H. Nakayama 1,2
1Department of Biomedical Engineering, Oregon Health & Science University, 2Department of Orthopaedics and Rehabilitation, Oregon Health & Science University

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

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