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Wayne State University School of Medicine

26 ARTICLES PUBLISHED IN JoVE

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Biology

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina
Ryan Thummel 1, Travis J. Bailey 2,3, David R. Hyde 2,3
1Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine, 2Department of Biological Sciences, University of Notre Dame , 3Center for Zebrafish Research, University of Notre Dame

A method to conditionally knockdown a target protein’s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein’s role in the regenerating or intact retina.

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Biology

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin
David R. Hyde 1, Alan R. Godwin 2, Ryan Thummel 3
1Department of Biological Sciences, Center for Zebrafish Research, University of Notre Dame , 2Department of Microbiology, Immunology, and Pathology, Colorado State University , 3Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein’s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

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Immunology and Infection

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge
Michael K. Shaw 1, Xiao-qing Zhao 2, Harley Y. Tse 2
1Department of Medicine, Section of Cardiology, St. John-Providence Health System, 2Department of Immunology and Microbiology, Wayne State University School of Medicine

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

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Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Yanning Wu 1, Shuo Wang 1, Chunying Li 1,2,3
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

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Medicine

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia
Thomas H. Sanderson 1,2,3, Joseph M. Wider 2,3
1Department of Emergency Medicine, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Department of Physiology, Wayne State University School of Medicine

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

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Behavior

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Evan D. Morris 1,2,3,4, Su Jin Kim 1,3, Jenna M. Sullivan 1,3,4, Shuo Wang 3,4, Marc D. Normandin 5, Cristian C. Constantinescu 6, Kelly P. Cosgrove 1,2,3
1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

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Behavior

Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice
Mariana Angoa-Pérez 1, Michael J. Kane 1, Denise I. Briggs 1, Dina M. Francescutti 1, Donald M. Kuhn 1
1Research and Development Service, John D. Dingell VA Medical Center and Department of Psychiatry & Behavioral Neurosciences, Wayne State University School of Medicine

Disease, head injury, genetic modifications, and treatment of mice with drugs can have profound effects on behavior. Utilizing well-characterized and validated approaches such as marble burying and nestlet shredding, compulsive-like behaviors can be documented accurately in mice as models of human obsessive-compulsive disorder and autism spectrum disorder.

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Neuroscience

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish
Jennifer L. Thomas 1, Ryan Thummel 1,2
1Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 2Department of Opthalmology, Wayne State University School of Medicine

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

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Biology

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor
Miquella G. Chavez 1,2, Jimmy Hu 1, Kerstin Seidel 1, Chunying Li 1,3, Andrew Jheon 1, Adrien Naveau 4,5,6, Orapin Horst 1,7, Ophir D. Klein 1,8
1Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology, University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 3Department of Pathology and Research Center, Zhongshan Hospital of Dalian University, 4Université Paris Descartes, Sorbonne Paris Cite, UMR S872, 5Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMR S872, 6INSERM U872, 7Division of Endodontics, Department of Preventive and Restorative Dental Sciences, University of California, San Francisco, 8Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

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Medicine

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting
Amy Weckle 1,2, Allison E. Aiello 3, Monica Uddin 1,4, Sandro Galea 5, Rebecca M. Coulborn 6, Richelo Soliven 7, Helen Meier 6, Derek E. Wildman 1,2
1Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 2Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 3Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, 4Department of Psychology, University of Illinois at Urbana-Champaign, 5Department of Epidemiology, Mailman School of Public Health, Columbia University, 6Department of Epidemiology, University of Michigan School of Public Health, 7Center for Molecular Medicine and Genetics, Wayne State University School of Medicine

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

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Neuroscience

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques
Chase B. Hellmer 1, Tomomi Ichinose 1,2
1Anatomy and Cell Biology, Wayne State University School of Medicine, 2Ophthalmology, Wayne State University School of Medicine

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

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Immunology and Infection

Isolation of Leukocytes from the Human Maternal-fetal Interface
Yi Xu 1, Olesya Plazyo 1, Roberto Romero 1,2,3,4, Sonia S. Hassan 1,5, Nardhy Gomez-Lopez 1,5,6
1Perinatology Research Branch, NICHD/NIH/DHHS, 2Department of Obstetrics and Gynecology, University of Michigan, 3Department of Epidemiology and Biostatistics, Michigan State University, 4Department of Molecular Obstetrics and Genetics, Wayne State University, 5Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 6Department of Immunology and Microbiology, Wayne State University School of Medicine

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

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Immunology and Infection

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface
Marcia Arenas-Hernandez 1, Elly N. Sanchez-Rodriguez 1, Tara N. Mial 1, Sarah A. Robertson 2, Nardhy Gomez-Lopez 1,3,4
1Department of Obstetrics & Gynecology, Wayne State University School of Medicine, 2School of Paediatrics and Reproductive Health, Research Centre for Reproductive Health, the Robinson Research Institute, The University of Adelaide, 3Department of Immunology & Microbiology, Wayne State University School of Medicine, 4Perinatology Research Branch, NICHD/NIH/DHHS

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

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Biology

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
Karthika Natarajan 1,2, Yi Xie 1,3, Takeo Nakanishi 4, Rebecca S. Moreci 5,6, Pancharatnam Jeyasuria 7, Arif Hussain 1,3,8,9, Douglas D. Ross 1,3,8,9,10,11
1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Pharmaceutical Sciences, University of Maryland School of Pharmacy, 3Baltimore VA Medical Center, 4Membrane Transport and Biopharmaceutics, School of Pharmaceutical Sciences, Kanazawa University, 5Obstetrics, Gynecology and Reproductive Science, University of Pittsburgh, 6Magee Women's Research Institute, 7Obstetrics, Gynecology, Perinatal Research Branch (NICHD), Wayne State University School of Medicine, 8Medicine, University of Maryland School of Medicine, 9Pathology, University of Maryland School of Medicine, 10Pharmacology, University of Maryland School of Medicine, 11Experimental Therapeutics, University of Maryland School of Medicine

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

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Medicine

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix
Daniel Xie 1, Donghong Ju 2, Cecilia Speyer 2, David Gorski 2,3, Mary A. Kosir 2,3
1Center for Molecular Medicine and Genetics (CMMG), Wayne State University School of Medicine, 2Department of Surgery, Wayne State University School of Medicine, 3Department of Oncology, Wayne State University School of Medicine

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

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Neuroscience

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging
Morven A. Cameron 1, Orsolya Kekesi 1,2, John W. Morley 1,2, Alba Bellot-Saez 1,2, Sindy Kueh 2, Paul Breen 1, André van Schaik 1, Jonathan Tapson 1, Yossi Buskila 1,2
1The MARCS Institute, Western Sydney University, 2School of Medicine, Western Sydney University

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for >24 h.

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Developmental Biology

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells
Benjamin L. Kidder 1,2
1Department of Oncology, Wayne State University School of Medicine, 2Karmanos Cancer Institute, Wayne State University School of Medicine

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

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Immunology and Infection

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model
Dhrubajyoti Nag 1, Kristie Mitchell 1, Paul Breen 1, Jeffrey H. Withey 1
1Department of Microbiology, Immunology, and Biochemistry, Wayne State University School of Medicine

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

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Bioengineering

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods
Baozhen Zhang 1, Zhilong Chen 1,2, Jinyu Han 1,3, Mengxia Li 1, Nihar R. Nayak 4, Xiujun Fan 1
1Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2College of Veterinary Medicine, Hunan Agricultural University, 3Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, College of Chemistry and Material Sciences, Heilongjiang University, 4Department of Obstetrics and Gynecology, Wayne State University School of Medicine

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

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Immunology and Infection

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis
Shirley Albano-Aluquin 1, Jozef Malysz 2, Michal Kidacki 3, Manohar Ratnam 4, Nancy J. Olsen 1
1Department of Medicine, Penn State University, 2Department of Pathology, Penn State University, 3Penn State College of Medicine, 4Department of Oncology, Wayne State University School of Medicine

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

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Behavior

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients
Shuo Wang 1, Nand Chandravadia 2, Adam N. Mamelak 2, Ueli Rutishauser 2,3,4
1Department of Chemical and Biomedical Engineering, and Rockefeller Neuroscience Institute, West Virginia University, 2Departments of Neurosurgery and Neurology, Cedars-Sinai Medical Center, 3Center for Neural Science and Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Division of Biology and Biological Engineering, California Institute of Technology

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

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Behavior

Using Looming Visual Stimuli to Evaluate Mouse Vision
Christina C. Koehler 1, Leo M. Hall 1, Chase B. Hellmer 1, Tomomi Ichinose 1
1Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine

To examine mouse vision, we conducted a looming test. Mice were placed in a large square arena with a monitor on its ceiling. The looming visual stimulus consistently evoked freezing or flight reactions in the mice.

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Cancer Research

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging
Nour Ghaddar 1,2, Shuo Wang 1, Véronique Michaud 1, Urszula Kazimierczak 1,3, Nicolas Ah-son 1, Antonis E. Koromilas 1,4
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 2Division of Experimental Medicine, Faculty of Medicine, McGill University, 3Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 4Gerald Bronfman Department of Oncology, Faculty of Medicine, McGill University

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

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Neuroscience

High-frequency Ultrasonography of the Digital and Palmar Nerve Branches of the Hand in Peripheral Nerve Diseases
Arthur Yan *1, Luke Geierman *2, Chris Andrianno 1, Amira Badr 3, Ryan Castoro 1,2,4
1Department of Physical Medicine and Rehabilitation, Wayne State University School of Medicine, Detroit Medical Center, 2Department of Neurology, Wayne State University School of Medicine, 3Department of Occupational Therapy, Wayne State University School of Medicine, 4Division of Neuromuscular Medicine, Department of Neurology, Wayne State University School of Medicine

The present protocol describes high-frequency neuromuscular ultrasonography of the digital and palmar branches of the median and ulnar nerve, which can aid in localizing peripheral nerve diseases and be adapted to evaluate digital nerve injuries.

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Cancer Research

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum
Terrence Wong 1, Roslyn Tedja 2, Hussein Chehade 2, Robert Morris 1, Ayesha B. Alvero 2, Gil Mor 2
1Department of Gynecologic Oncology, Karmanos Cancer Institute, 2C.S. Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University School of Medicine

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

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Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing
Nan Gao 1, Mark McDermott 1, Fu-Shin Yu 1
1Departments of Ophthalmology and Anatomy and Cell Biology, Kresge Eye Institute, Wayne State University School of Medicine

Porcine corneal ex vivo organ culture and epithelial wound healing provide an economical, ethical, reproducible, and quantitative means for testing the ocular toxicity of chemicals. They also aid in elucidating mechanisms underlying the regulation of epithelialization and tissue repair, and in evaluating therapeutics for treating diabetic keratopathy and delayed wound healing.

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