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University of Missouri

47 ARTICLES PUBLISHED IN JoVE

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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

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation
Pooneh Bagher 1, Steven S. Segal 1,2
1Department of Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center, University of Missouri

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

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Biology

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation
Pooneh Bagher 1, Luis Polo-Parada 1,2, Steven S. Segal 1,2
1Department of Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center, University of Missouri

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

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Medicine

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice
Jacqueline J. Glascock 1, Erkan Y. Osman 1, Tristan H. Coady 2, Ferrill F. Rose 1, Monir Shababi 3, Christian L. Lorson 3
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University , 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

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Biology

Drosophila Pupal Abdomen Immunohistochemistry
Wei Wang 1, John H. Yoder 1
1Department of Biological Sciences, University of Alabama

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

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Biology

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay
Kathleen A. Pennington 1, Jessica M. Schlitt 1, Laura C. Schulz 1
1Obstetrics, Gynecology and Women's Health, University of Missouri

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

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Medicine

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue
Katherine E. Pelch 1, Kathy L. Sharpe-Timms 2, Susan C. Nagel 1
1Obstetrics, Gynecology and Women’s Health and Division of Biological Sciences, University of Missouri, 2Obstetrics, Gynecology and Women’s Health and Animal Sciences, University of Missouri

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

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Bioengineering

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry
Christine M. O'Brien 1, Kyle Rood 1, Shramik Sengupta 1, Sagar K. Gupta 1, Thiago DeSouza 1, Aaron Cook 1, John A. Viator 1
1Biological Engineering, Christopher S. Bond Life Sciences Center, Dermatology, University of Missouri

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

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Biology

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
Garrett M. Dahm 1,2, Matthew M. Gubin 1,2, Joseph D. Magee 2, Patsharaporn Techasintana 1,2, Robert Calaluce 2, Ulus Atasoy 1,2,3
1Molecular Microbiology and Immunology, University of Missouri, 2Department of Surgery, University of Missouri, 3Child Health, University of Missouri

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

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Bioengineering

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents
Carol E. Soteropulos 1, Heather K. Hunt 1
1Department of Biological Engineering, University of Missouri

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

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Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window
Lifeng Zhang *1, Andree Lapierre *1, Brittany Roy 1, Maili Lim 1, Jennifer Zhu 1, Wei Wang 1, Stephen B. Sampson 1, Kyuson Yun 1, Bonnie Lyons 1, Yun Li 1, Da-Ting Lin 1
1The Jackson Laboratory

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

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Neuroscience

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
Yun Li 1, Brittany D. Roy 1, Wei Wang 1, Lifeng Zhang 1, Stephen B. Sampson 1, Da-Ting Lin 1
1The Jackson Laboratory

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

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Biology

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice
Chady H. Hakim 1, Nalinda B. Wasala 1, Dongsheng Duan 1
1Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

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Biology

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries
Matthew J. Socha 1, Steven S. Segal 1,2
1Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

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Behavior

Barnes Maze Testing Strategies with Small and Large Rodent Models
Cheryl S. Rosenfeld *1, Sherry A. Ferguson *2
1Biomedical Sciences and Bond Life Sciences Center, University of Missouri, 2Division of Neurotoxicology, National Center for Toxicological Research, Food and Drug Administration

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g. latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

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Medicine

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models
Teresa E. Lever 1, Sabrina M. Braun 2, Ryan T. Brooks 2, Rebecca A. Harris 2, Loren L. Littrell 2, Ryan M. Neff 3, Cameron J. Hinkel 3, Mitchell J. Allen 1, Mollie A. Ulsas 2
1Department of Otolaryngology - Head and Neck Surgery, University of Missouri, 2Department of Communication Science and Disorders, University of Missouri, 3Department of Medicine, University of Missouri

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

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Chemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization
Wei Wang 1, John M. Lester 1, Anthony E. Amorosa 2, Deborah L. Chance 3, Valeri V. Mossine 1, Thomas P. Mawhinney 1,4
1Department of Biochemistry, University of Missouri, 2Department of Biological Sciences, University of Missouri, 3Department of Molecular Microbiology & Immunology, University of Missouri, 4Department of Child Health, University of Missouri

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

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Medicine

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice
Hailong Li *1, Gourav Roy Choudhury *1, Nannan Zhang 1, Shinghua Ding 1
1Department of Bioengineering, Dalton Cardiovascular Research Center, University of Missouri

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

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Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons
Hailong Li *1, Marina Aksenova *1, Sarah J. Bertrand 1, Charles F. Mactutus 1, Rosemarie Booze 1
1Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

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Engineering

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces
Benjamin S. Goldschmidt 1, Anna M. Rudy 2, Charissa A. Nowak 3, Yowting Tsay 3, Paul J. D. Whiteside 3, Heather K. Hunt 3
1Biomedical Engineering, Duquesne University, 2Department of Computer Science, University of Missouri, 3Department of Bioengineering, University of Missouri

Here we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. This technique evanescent field-based photoacoustics can be used to create a photoacoustic metrology system to estimate materials' thicknesses, bulk and thin film refractive indices, and explore their optical properties.

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Near-infrared Navigation System for Real-time Visualization of Blood Flow in Vascular Grafts
Srikar Raman *1, Sarah Hansen *2, Charles Jr. Caldwell 3, Vaishnavi Raman 1, Henry White 1, Craig Emter 4, Ajit Tharakan 5, Anandhi Upendran 6,7, Raghuraman Kannan 1,3
1Department of Radiology, University of Missouri, 2Office of Animal Resources, University of Missouri, 3Bioengineering, University of Missouri, 4Biomedical Science, University of Missouri, 5Surgery - Division of Cardiothoracic Surgery, University of Missouri, 6MU-iCATS, University of Missouri, 7Medical Pharmacology and Physiology, University of Missouri

We describe the procedure for real-time monitoring of blood flow in vascular grafts using indocyanine green (ICG), a near-infrared (NIR) dye, and a portable near-infrared navigation (NAVI) detectible camera system. The flow of dye in vascular grafts and the camera efficiency have been compared with Doppler and cine-angiography procedures.

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Engineering

Precision Milling of Carbon Nanotube Forests Using Low Pressure Scanning Electron Microscopy
Josef Brown 1, Benjamin F. Davis 1, Matthew R. Maschmann 1
1Department of Mechanical & Aerospace Engineering, University of Missouri

Low pressure scanning electron microscopy in a water vapor ambient is used to machine nanoscale to microscale features in carbon nanotube forests.

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Biology

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth
Matthew Howell 1, Jeremy J. Daniel 1, Pamela J.B. Brown 1
1Division of Biological Sciences, University of Missouri

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

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Neuroscience

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology
Hailong Li 1, Jessica M. Illenberger 1, Kristen A. McLaurin 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

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Medicine

A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts
John Harry Caufield 1,2, David A. Liem 1,2,3, Anders O. Garlid 1,2, Yijiang Zhou 4, Karol Watson 1,3, Alex A. T. Bui 1,5,6,7, Wei Wang 1,7,8,9, Peipei Ping 1,2,3,7,8
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Medicine/Cardiology, University of California, Los Angeles, 4Department of Cardiology, First Affiliated Hospital, Zhejiang University School of Medicine, 5Department of Radiological Sciences, University of California, Los Angeles, 6Department of Bioengineering, University of California, Los Angeles, 7Scalable Analytics Institute (ScAi), University of California, Los Angeles, 8Department of Bioinformatics, University of California, Los Angeles, 9Department of Computer Science, University of California, Los Angeles

We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

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Neuroscience

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents
Kristen A McLaurin 1, Landhing M Moran 1, Hailong Li 1, Rosemarie M Booze 1, Charles F Mactutus 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

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Medicine

Cloud-Based Phrase Mining and Analysis of User-Defined Phrase-Category Association in Biomedical Publications
Dibakar Sigdel *1,2, Vincent Kyi *1,2, Aiden Zhang *1, Shaun P. Setty 3, David A. Liem 1,2,4, Yu Shi 5, Xuan Wang 5, Jiaming Shen 5, Wei Wang 1,6,7, JiaWei Han 5, Peipei Ping 1,2,4,6
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Pediatric and Adult Congenital Heart Surgery, Miller Children's and Women's Hospital and Long Beach Memorial Hospital, 4Department of Medicine/Cardiology, University of California, Los Angeles, 5NIH BD2K Program Centers of Excellence for Big Data Computing -- KnowEng Center, Department of Computer Science, University of Illinois at Urbana-Champaign (UIUC), 6Scalable Analytics Institute (ScAi), University of California, Los Angeles, 7Department of Computer Science, University of California, Los Angeles

We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain.

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Medicine

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach
Ali R. Ahmadi 1, Le Qi 1, Kenichi Iwasaki 1, Wei Wang 1, Russell N. Wesson 1, Andrew M. Cameron 1, Zhaoli Sun 1
1Department of Surgery, Johns Hopkins University School of Medicine

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

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Environment

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System
Weitao Hou 1, Hanpeng Wang 1, Wei Wang 1, Zhongzhong Liu 1, Qingchuan Li 1
1Research Center of Geotechnical and Structural Engineering, Shandong University

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal’s physical and mechanical properties induced by CO2 adsorption.

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Environment

Using Deuterium Oxide as a Non-Invasive, Non-Lethal Tool for Assessing Body Composition and Water Consumption in Mammals
Sarah E. Hooper 1, Amanda N. Eshelman 2, Ashley N. Cowan 3, Alicia Roistacher 4, Tyler S. Paneitz 2, Sybill K. Amelon 5
1Department of Veterinary Pathobiology, University of Missouri, 2College of Veterinary Medicine, University of Missouri, 3Department of Animal Science, University of Missouri, 4School of Natural Resources, University of Missouri, 5USDA USFS Northern Research Station

This article describes the deuterium oxide dilution technique in two mammals, an insectivore and carnivore, to determine total body water, lean body mass, body fat mass, and water consumption.

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JoVE Core

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation
Emily A. Brown 1,2, Steven C. Neier 3,4, Claudia Neuhauser 5, Adam G. Schrum 6,7,8, Stephen E.P. Smith 1,2,9
1Center for Integrative Brain Research, Seattle Children's Research Institute, 2Graduate Program in Neuroscience, University of Washington, 3Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 4Broad Institute of Harvard and MIT, 5Department of Mathematics, University of Houston, 6Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, 7Department of Surgery, School of Medicine, University of Missouri, 8Department Bioengineering, College of Engineering, University of Missouri, 9Department of Pediatrics, University of Washington

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

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Medicine

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Di Liu *1, Xizhu Xu *2, Yuejin Li 2, Jie Zhang 1, Xiaoyu Zhang 1, Qihuan Li 1, Haifeng Hou 2, Dong Li 2, Wei Wang 1,2,3, Youxin Wang 1
1Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, 2School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, 3School of Medical and Health Sciences, Edith Cowan University

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

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Immunology and Infection

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model
Neekun Sharma *1,3, Zhe Sun *2,3, Michael A. Hill 1,2,3, Chetan P. Hans 1,2,3
1Division of Cardiovascular Medicine, University of Missouri, 2Medical Pharmacology and Physiology, University of Missouri, 3Dalton Cardiovascular Research Center, University of Missouri

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

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Neuroscience

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture
Hailong Li 1, Kristen A. McLaurin 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

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Behavior

Loneliness Assuaged: Eye-Tracking an Audience Watching Barrage Videos
Guangyao Chen 1, Shuhua Zhou 2
1School of Journalism & Communication/National Media & Experimental Teaching Center, Jinan University, 2School of Journalism, University of Missouri

The study proposes an activation-match model to study how loneliness is mitigated when a lonely audience watches barrage videos of rational and emotional appeals. The protocol uses eye tracking to document duration and fixation, accounting for the degree of satisfaction when emotional needs are appeased by content and barrage.

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Behavior

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice
Yi Xie 1, Sai-Yue Deng 1, Si-Miao Chen 1, Xue-Jiao Chen 1, Wen-Wen Lai 1, Li-Fang Huang 1, Li Ba 1, Wei Wang 1,2, Feng-Fei Ding 1,3
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China, 2Key Laboratory of Neurological Diseases of Chinese Ministry of Education, The School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000,China, 3Department of Pharmacology, School of Basic Medical Science; State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

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Neuroscience

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue
Kristin N. Kirchner 1, Hailong Li 1, Adam R. Denton 1, Steven B. Harrod 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

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Neuroscience

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method
Weiyan Jiang *1, Congcong Jin *2, Weiming Xu *3, Yingxian Li 4, Yinghong Lin 5, Shengxiang Liang 6,7, Wei Wang 8
1Department of Clinical Laboratory, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 2Rehabilitation Department, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, 3Department of Neurosurgery, the Hospital of Changle, 4Pediatrics Ward 2, Shouguang Hospital of Traditional Chinese Medicine, 5Fujian University of Traditional Chinese Medicine, 6National-Local Joint Engineering Research Center of Rehabilitation Medicine Technology, Fujian University of Traditional Chinese Medicine, 7Rehabilitation Industry Institute, Fujian University of Traditional Chinese Medicine, 8Department of Neurosurgery, the First Affiliated Hospital of Wenzhou Medical University

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

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Neuroscience

A Rat Model of EcoHIV Brain Infection
Hailong Li 1, Kristen A. McLaurin 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

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Neuroscience

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)
Nannan Zhang 1, Zhe Zhang 1,2, Ilker Ozden 2, Shinghua Ding 1,2
1Dalton Cardiovascular Research Center, University of Missouri-Columbia, 2Department of Biomedical, Biological and Chemical Engineering, University of Missouri-Columbia

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

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Developmental Biology

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes
Daniela Londoño-Vásquez 1,2, Alexander Jurkevich 3, Ahmed Z. Balboula 1
1Animal Sciences Research Center, University of Missouri, 2Universidad Nacional de Colombia, 3Advanced Light Microscopy Core, University of Missouri

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

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Biology

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging
Janlo M. Robil 1,2, Cedric David T. Cortez 1, Chloe Margalaux R. Villafuerte 1, Edgar Daniel C. Dela Peña 1, Derreck O. De Leon 1, Roselle Joy R. Rioja 1, Paula McSteen 2
1Department of Biology, School of Science and Engineering, Ateneo de Manila University, 2Division of Biological Sciences, Interdisciplinary Plant Group, and Missouri Maize Center, University of Missouri

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

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Bioengineering

Troubleshooting and Quality Assurance in Hyperpolarized Xenon Magnetic Resonance Imaging: Tools for High-Quality Image Acquisition
Ummul Afia Shammi 1, Gabriela María Garcίa Delgado 2, Robert Thomen 2,3
1Roy Blunt NextGen Precision Health Center, University of Missouri, 2Chemical and Biomedical Engineering, University of Missouri, 3Radiology, School of Medicine, University of Missouri

Here, we present a protocol for obtaining high-quality hyperpolarized xenon-129 magnetic resonance images, covering hardware, software, data acquisition, sequence selection, data management, k-space utilization, and noise analysis.

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Genetics

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation
Daniel J. Davis 1,2,4, James F. McNew 2, Jennifer N. Walls 3, Christine E. Bethune 3, Payton S. Oswalt 3, Elizabeth C. Bryda 1,2,3,4
1Animal Modeling Core, University of Missouri, 2Comparative Medicine Program, University of Missouri, 3Rat Resource and Research Center, University of Missouri, 4Department of Veterinary Pathobiology, University of Missouri

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

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Behavior

Examining Gesture Production in the Presence of Communication Challenges
Laura M. Morett 1
1Department of Speech, Language, and Hearing Sciences, University of Missouri

Here, we present a protocol that manipulates interlocutor visibility to examine its impact on gesture production in interpersonal communication. This protocol is flexible to tasks implemented, gestures examined, and communication modality. It is ideal for populations with communication challenges, such as second language learners and individuals with autism spectrum disorder.

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Neuroscience

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models
Arsh Ketabforoush 1, Meifang Wang 1, W. David Arnold 1,2,3,4
1NextGen Precision Health, University of Missouri, 2Department of Physical Medicine and Rehabilitation, University of Missouri, 3Department of Neurology, University of Missouri, 4Department of Medical Pharmacology and Physiology, University of Missouri

In this study, we demonstrate a refined single fiber electromyography (SFEMG) protocol to allow in vivo measurement of neuromuscular junction (NMJ) transmission in rodent models. A step-by-step approach to the SFEMG technique is described to allow quantification of NMJ transmission variability and failure in rat gastrocnemius muscle.

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Neuroscience

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation
Arsh Ketabforoush *1, Meifang Wang *1, Catherine L. Smith 2, William David Arnold 1,3,4,5, Nicole L. Nichols 2,5,6
1NextGen Precision Health, University of Missouri, 2Department of Biomedical Sciences, University of Missouri, 3Department of Physical Medicine and Rehabilitation, University of Missouri, 4Department of Neurology, University of Missouri, 5Department of Medical Pharmacology and Physiology, University of Missouri, 6Dalton Cardiovascular Research Center, University of Missouri

In this study, we present an in vivo method for estimating motor unit number and size to quantify rat diaphragm motor unit connectivity. A step-by-step approach to these techniques is described.

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