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Indiana University School of Medicine

57 ARTICLES PUBLISHED IN JoVE

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Medicine

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart
Surya C. Gnyawali 1,2, Sashwati Roy 1,2, Jason Driggs 1,2, Savita Khanna 1,2, Thomas Ryan 2,3, Chandan K. Sen 2
1Department of Surgery, The Ohio State University, 2Heart and Lung Research Institute, The Ohio State University, 3Department of Cardiovascular Medicine, The Ohio State University

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

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Biology

Isolating Stem Cells from Soft Musculoskeletal Tissues
Yong Li 1,2,3,4, Haiying Pan 1, Johnny Huard 1,2,3,4,5
1Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, 2Department of Bioengineering, University of Pittsburgh, 3Department of Orthopedic Surgery, University of Pittsburgh, 4Department of Pathology, University of Pittsburgh, 5Department of Molecular Genetics & Biochemistry, University of Pittsburgh

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

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Medicine

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function
Ronald X. Xu 1, Kun Huang 2, Ruogu Qin 1, Jiwei Huang 1, Jeff S. Xu 1, Liya Ding 2, Urmila S. Gnyawali 3, Gayle M. Gordillo 3, Surya C. Gnyawali 3,4, Chandan K. Sen 3
1Department of Biomedical Engineering, The Ohio State University, 2Department of Biomedical Informatics, The Ohio State University, 3Comprehensive Wound Center, The Ohio State University, 4Department of Surgery, The Ohio State University

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

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Neuroscience

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents
Wenhui Xiong 1, Xingjie Ping 1, Jianhua Gao 1, Xiaoming Jin 1
1Department of Anatomy and Cell Biology, Department of Neurosurgery, Stark Neuroscience Research Institute, Indiana University School of Medicine

Partially isolated cortex (“undercut”) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

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Medicine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice
Julio E. Ayala 1, Deanna P. Bracy 2,3, Carlo Malabanan 3, Freyja D. James 2,3, Tasneem Ansari 3, Patrick T. Fueger 4, Owen P. McGuinness 2,3, David H. Wasserman 2,3
1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

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Biology

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood
Nutan Prasain 1, J. Luke Meador 1, Mervin C. Yoder 1
1Herman B Wells Center for Pediatric Research, Indiana University School of Medicine

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

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Medicine

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation
Hidemi Suzuki 1,2, Lin Fan 1,2, David S. Wilkes 1,2
1Departments of Medicine, Microbiology and Immunology, Indiana University School of Medicine, 2Center for Immunobiology, Indiana University School of Medicine

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

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Medicine

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Patrick T. Fueger 1,2, Angelina M. Hernandez 2, Yi-Chun Chen 2, E. Scott Colvin 1,2
1Department of Pediatrics, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

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Biology

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
Natalie D. Stull 1, Andrew Breite 2, Robert McCarthy 2, Sarah A. Tersey 1, Raghavendra G. Mirmira 1,3,4
1Department of Pediatrics and the Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 2VITACYTE, LLC, 3Department of Medicine, Indiana University School of Medicine, 4Department of Cellular and Integrative Physiology, Indiana University School of Medicine

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

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Medicine

Controlled Cervical Laceration Injury in Mice
Yi Ping Zhang 1, Melissa J. Walker 2, Lisa B. E. Shields 1, Xiaofei Wang 2, Chandler L. Walker 2, Xiao-Ming Xu 2, Christopher B. Shields 1
1Norton Neuroscience Institute, Norton Healthcare, 2Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Medical Neuroscience Graduate Program, and Department of Anatomy and Cell Biology, Indiana University School of Medicine

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm.

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Medicine

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats
Ruben M. Sandoval 1, Bruce A. Molitoris 1
1Medicine/Nephrology, Indiana University School of Medicine

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

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Medicine

A Novel Vertebral Stabilization Method for Producing Contusive Spinal Cord Injury
Melissa J. Walker *1,2, Chandler L. Walker *1,3, Y. Ping Zhang 4, Lisa B. E. Shields 4, Christopher B. Shields 4, Xiao-Ming Xu 1,3
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Indiana University School of Medicine, 2Medical Neuroscience Graduate Program, Indiana University School of Medicine, 3Department of Anatomy and Cell Biology, Indiana University School of Medicine, 4Norton Neuroscience Institute, Norton Healthcare

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.  Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

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Biology

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue
Morgan H. McCoy 1, Kristin Post 2, Joyashree D. Sen 2, Hsim Y. Chang 2, Zijin Zhao 1, Rong Fan 1, Shaoxiong Chen 1, Diane Leland 1, Liang Cheng 1, Jingmei Lin 1
1Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 2Department of Pathology and Laboratory Medicine, Indiana University Health

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

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Medicine

Controlled Cortical Impact Model for Traumatic Brain Injury
Jennifer Romine 1, Xiang Gao 1, Jinhui Chen 1
1Spinal Cord and Brain Injury Research Group, Stark Neuroscience Research Institute, Department of Neurological Surgery, Indiana University School of Medicine

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

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Medicine

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines
Jennifer M. Cole 1, Stancy Joseph 1, Christopher G. Sudhahar 1, Karen D. Cowden Dahl 1
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine

Cancer stem cells (CSCs) are implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing in a stem cell promoting media we enrich for non-adherent CSC cultures.

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Biology

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation
Emmanuel Bikorimana 1,2, Danica Lapid 3, Hyewon Choi 3, Richard Dahl 1,2,3
1Harper Cancer Research Institute, 2Microbiology and Immunology, Indiana University School of Medicine, 3Department of Biological Sciences, University of Notre Dame

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

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Medicine

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury
Deborah N. Olmstead 1,2, Nichole A. Mesnard-Hoaglin 3, Richard J. Batka 1,2, Melissa M. Haulcomb 1,2, Whitney M. Miller 1,2, Kathryn J. Jones 1,2
1Anatomy and Cell Biology, Indiana University School of Medicine, 2Research and Development Services, Richard L. Roudebush VA Medical Center, 3Department of Anatomy and Cell Biology, University of Illinois, Chicago

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

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Biology

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae
Xin Zhang *1, Keshava Mysore *2,3, Ellen Flannery 3,4, Kristin Michel 1, David W. Severson 3,4, Kun Yan Zhu 5, Molly Duman-Scheel 2,3,4
1Division of Biology, Kansas State University, 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, 3Eck Institute for Global Health, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame, 5Department of Entomology, Kansas State University

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

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Developmental Biology

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches
Lin Wang *1, Malgorzata M. Kamocka *2, Amy Zollman 3, Nadia Carlesso 3
1Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, 2Indiana Center for Biological Microscopy, Department of Medicine, Indiana University School of Medicine, 3Department of Pediatrics, Indiana University School of Medicine

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

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Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
Nathan J. Alves 1,2, Kendrick B. Turner 1, Scott A. Walper 1
1Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, 2Department of Emergency Medicine, Indiana University of School of Medicine

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

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Genetics

Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death
Sarah A Tersey 1, Jennifer B Nelson 1, Marisa M Fisher 2, Raghavendra G Mirmira 1,3,4
1Department of Pediatrics, IU Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, 2Department of Pediartics, Omaha Children's Hospital and Medical Center, University of Nebraska Medical Center, 3Departments of Biochemistry and Molecular Biology, Medicine, and Cellular and Integrative Physiology, IU Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, 4Indiana Biosciences Research Institute

Islet β cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of β cell death.

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Cancer Research

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia
Andrea Bonetto 1, Joseph E. Rupert 1, Rafael Barreto 1, Teresa A. Zimmers 1
1Department of Surgery, Simon Cancer Center and IUPUI Center for Cachexia Research, Innovation and Therapy, Indiana University School of Medicine

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

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Medicine

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice
Xiangbing Wu *1, Yi Ping Zhang *2, Wenrui Qu 1, Lisa B. E. Shields 2, Christopher B. Shields 2,3, Xiao-Ming Xu 1
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Indiana University School of Medicine, 2Norton Neuroscience Institute, Norton Healthcare, 3Department of Anatomical Sciences and Neurobiology, University of Louisville

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

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Neuroscience

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice
Xiaojing Lin 1,2, Tingbao Zhao 3, Wenhui Xiong 1, Shaonan Wen 4, Xiaoming Jin 1, Xiao-Ming Xu 1
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Department of Anatomy and Cell Biology, Indiana University School of Medicine, 2Department of Spinal Cord Injury and Repair, Trauma and Orthopedics Institute of Chinese PLA, General Hospital of Jinan Military Region, 3Department of Orthopedics, Shandong Cancer Hospital, Shandong University, 4Department of Neurobiology, Institute of Basic Medical Sciences, Academy Military Medical Sciences

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

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Neuroscience

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury
Chen Chen 1,2, Yi Ping Zhang 3, Yan Sun 1,4, Wenhui Xiong 1, Lisa B. E. Shields 3, Christopher B. Shields 3,5, Xiaoming Jin 1, Xiao-Ming Xu 1
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, and Department of Neurological Surgery, Indiana University School of Medicine, 2Program in Medical Neuroscience, Stark Neurosciences Research Institute, Indiana University School of Medicine, 3Norton Neuroscience Institute, Norton Healthcare, 4Department of Human Anatomy & Histoembryology, School of Basic Medical Sciences, Fudan University, 5Department of Neurological Surgery, University of Louisville School of Medicine

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

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Developmental Biology

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro
Kaela M. Varberg 1,2, Seth Winfree 3,4, Kenneth W. Dunn 3,4, Laura S. Haneline 1,2,5,6,7
1Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 2Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 3Indiana Center for Biological Microscopy, Indiana University School of Medicine, 4Department of Medicine, Indiana University School of Medicine, 5Department of Pediatrics, Indiana University School of Medicine, 6Department of Microbiology and Immunology, Indiana University School of Medicine, 7Indiana University Simon Cancer Center, Indiana University School of Medicine

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

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Medicine

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats
Xiao-Jing Lin 1,2,3, Shaonan Wen 2,6, Ling-Xiao Deng 3, Heqiao Dai 3, Xiaolong Du 3, Chen Chen 3,4, Melissa J. Walker 3,4, Ting-Bao Zhao 1,5, Xiao-Ming Xu 3
1Department of Spinal Cord Injury and Repair, Trauma and Orthopedics Institute of Chinese PLA, 960th Hospital, Joint Logistics Support Force of PLA, 2Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Academy of Military Sciences, 3Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman Campbell Brain and Spine, Indiana University School of Medicine, 4Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, 5Department of Orthopedics, the Third Affiliated Hospital, Shandong University, 6Beijing Computing Center, Beijing Academy of Science and Technology

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

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Medicine

Mechanical Ventilation Boot Camp Curriculum
Jennifer Yee 1,2, Alma Benner 2, Jared Hammond 2, Bethany Malone 2, Charles Fuenning 3, Richard George 2,4, Rami A. Ahmed 2,3,4
1Department of Emergency Medicine, The Ohio State University, 2Summa Health System, Akron City Hospital, 3Western Reserve Hospital, 4Northeast Ohio Medical University

This curriculum outlines how to execute a simulation-based boot camp to teach providers how to manage mechanically ventilated patients.

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Medicine

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

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

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JoVE Core

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing
Justin N. Williams 1, Yong Li 1, Anuradha Valiya Kambrath 1, Uma Sankar 1
1Department of Anatomy and Cell Biology, Indiana University School of Medicine

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

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Biology

A Proximal Culture Method to Study Paracrine Signaling Between Cells
Subramanyam Dasari 1, Taruni Pandhiri 1, James Haley 1, Dean Lenz 2, Anirban K. Mitra 1,3,4
1Indiana University School of Medicine, 2Urology, Indiana University Health Southern Indiana Physicians, 3Indiana University Melvin and Bren Simon Cancer Center, 4Department of Medical and Molecular Genetics, Indiana University School of Medicine

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

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Chemistry

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
Elijah N. McCool *1, Rachele Lubeckyj *1, Xiaojing Shen 1, Qiang Kou 2, Xiaowen Liu 2,3, Liangliang Sun 1
1Department of Chemistry, Michigan State University, 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 3Center for Computational Biology and Bioinformatics, Indiana University School of Medicine

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

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Biochemistry

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation
Ying Fan *1, Francesca Tonelli *1, Shalini Padmanabhan 2, Marco A.S. Baptista 2, Lindsey Riley 2, Danielle Smith 3, Connie Marras 4, Andrew Howden 5, Dario R. Alessi 1, Esther Sammler 1,6
1MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2The Michael J. Fox Foundation for Parkinson's Research, 3Department of Medical and Molecular Genetics, Indiana University School of Medicine, 4Morton and Gloria Shulman Movement Disorders Centre and the Edmond J. Safra Program in Parkinson's Disease, Toronto Western Hospital, University of Toronto, 5Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, 6Division of Molecular and Clinical Medicine, School of Medicine, Ninewells Hospital and Medical School, University of Dundee

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

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JoVE Core

Setup and Execution Of the Blindfolded Code Training Exercise
Patrick G. Hughes 1, Kate E. Hughes 2, Rami A. Ahmed 3
1Florida Atlantic University Schmidt College of Medicine, 2Department of Emergency Medicine, University of Arizona College of Medicine, 3Department of Emergency Medicine, Indiana University School of Medicine

The blindfolded code training exercise, which blindfolds the team leader in a code resuscitation simulation, is an advanced teaching technique to improve closed-loop communication, organizational skills, and critical thinking. 

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Biology

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis
Kaley L. Liang *1, Julia O. Bursova *1, Frank Lam 1, Xingjuan Chen 1, Alexander G. Obukhov 1
1The Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

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Immunology and Infection

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice
Ming Meng *1,2, Shengde Chen *1,2, Xiang Gao *1,2, Huifang Liu *1,2, Yuanyuan Wang *1,2, Jingnan Zhang *1,2, Haiyang Dou *1,2, Wenjuan Li *1,2, Dongzhi Chen *1,2
1Medical School of Hebei University, 2Key Laboratory of Pathogenesis Mechanism and Control of Inflammatory-Autoimmune Diseases in Hebei Province

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

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Bioengineering

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature
Sherry G. Clendenon 1,2, Xiao Fu 1,3, Robert A. Von Hoene 1, Jeffrey L. Clendenon 4, James P. Sluka 1,2, Seth Winfree 5, Henry Mang 5, Michelle Martinez 5, Adele Filson 5, James E. Klaunig 6, James A. Glazier 1,2, Kenneth W. Dunn 5
1Biocomplexity Institute, Indiana University, 2Department of Intelligent Systems Engineering, Indiana University, 3Department of Physics, Indiana University, 4Scientific Designs, 5Department of Medicine, Indiana University, 6School of Public Health, Indiana University

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

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Biology

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue
Daria Barwinska 1, Michael J. Ferkowicz 1, Ying-Hua Cheng 1, Seth Winfree 1,2, Kenneth W. Dunn 1, Katherine J. Kelly 1, Timothy A. Sutton 1, Brad H. Rovin 3, Samir V. Parikh 3, Carrie L. Phillips 4, Pierre C. Dagher 1, Tarek M. El-Achkar 1, Michael T. Eadon 1,
1Department of Medicine, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 3Division of Nephrology, Department of Medicine, Ohio State University Wexner Medical Center, 4Division of Pathology, Indiana University School of Medicine

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

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Medicine

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization
Ziqian Zeng 1,2, Tanmaye Nallan Chakravarthula 1,2, Nathan J. Alves 1,2
1Emergency Medicine Department, Indiana University School of Medicine, 2Weldon School of Biomedical Engineering, Purdue University

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

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Neuroscience

Evaluating Postural Control and Lower-extremity Muscle Activation in Individuals with Chronic Ankle Instability
Lulu Yin 1, Zhangqi Lai 2, Xiaoyue Hu 2, Kun Liu 3, Lin Wang 2
1Department of Critical Care Medicine, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, 2School of Kinesiology, Shanghai University of Sport, 3Department of Rehabilitation Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Individuals with chronic ankle instability (CAI) exhibit postural control deficiency and delayed muscle activation of lower extremities. Computerized dynamic posturography combined with surface electromyography provides insights into the coordination of the visual, somatosensory, and vestibular systems with muscle activation regulation to maintain postural stability in individuals with CAI.

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JoVE Core

Setup and Execution of the Rapid Cycle Deliberate Practice Death Notification Curriculum
Patrick G. Hughes 1, Kate E. Hughes 2, Mary J. Hughes 3,4, Lindsay Weaver 4, Lauren E. Falvo 4, Anna M. Bona 4, Dylan Cooper 4, Cherri Hobgood 4, Rami A. Ahmed 4
1Florida Atlantic University Schmidt College of Medicine, 2Department of Emergency Medicine, University of Arizona College of Medicine, 3Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, 4Indiana University School of Medicine

The goal is to demonstrate how to apply the rapid cycle deliberate practice debriefing technique to the GRIEV_ING death notification curriculum.

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Behavior

The Participant-Reported Implementation Update and Score (PRIUS): A Novel Method for Capturing Implementation-Related Data Over Time
Edward J. Miech 1,2,3,4,5, Nicholas A. Rattray 1,2,3,6, Dawn M. Bravata 1,2,3,5, Jennifer Myers 1,3, Teresa M. Damush 1,2,3,5
1VA Precision Monitoring (PRIS-M) QUERI, Richard L. Roudebush VA Medical Center, 2William M. Tierney Center for Health Services Research, Regenstrief Institute, 3VA Health Services Research & Development Center for Health Information and Communication (CHIC), Richard L. Roudebush VA Medical Center, 4Department of Emergency Medicine, Indiana University School of Medicine, 5Department of Internal Medicine and Geriatrics, Indiana University School of Medicine, 6Department of Anthropology, Indiana University-Purdue University Indianapolis

This protocol describes a novel method for collecting and analyzing data related to ongoing implementation called the Participant-Reported Implementation Update and Score (PRIUS). The PRIUS method allows for the efficient and systematic capture of data over time and from multiple viewpoints in healthcare settings.

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Medicine

A Murine Tail Lymphedema Model
Aladdin H. Hassanein *1, Mithun Sinha *1, Colby R. Neumann 1, Ganesh Mohan 1, Imran Khan 1, Chandan K. Sen 1
1Department of Surgery, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine

Lymphedema is extremity swelling caused by lymphatic dysfunction. We describe a chronic murine tail model of lymphedema and the novel use of tissue nanotransfection technology (TNT) for genetic cargo delivery to the tail.

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Engineering

Measurement of the Hand Transmitted Vibration of the Human Hand Arm System During Operation of a Hand Tractor
Shiqing Lu 1, Rui Jiang 2, Xinyi Xiao 3, Yong Li 1, Xia Huang 1, Kun Song 1, Changming Chen 1, Jun Ding 1
1School of Mechanical Engineering, Chongqing University of Technology, 2College of Mechanical and Electrical Engineering, Wenzhou University, 3Mechanical and Manufacturing Engineering, Miami University

Here, we present a standardized method for measurement of the hand transmitted vibration from handles of a single-axle tractor with special reference to changes in grip force and vibration frequency.

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Biology

Artificial Intelligence Approaches to Assessing Primary Cilia
Ruchi Bansal 1, Staci E. Engle 1, Tisianna K. Kamba 1, Kathryn M. Brewer 1, Wesley R. Lewis 2, Nicolas F. Berbari 1,3,4
1Department of Biology, Indiana University-Purdue University Indianapolis, 2Nikon Instruments Inc., 3Stark Neurosciences Research Institute, Indiana University, 4Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

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Biology

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence
Cassandra Warden *1, Alan J. Simmons *2, Lejla Pasic 3, Ashley Pitzer 4,6, Sean S. Davies 4, Justin H. Layer 5, Raymond L. Mernaugh 3, Annet Kirabo 4,6
1Vanderbilt Eye Institute, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Department of Biochemistry, Vanderbilt University, 4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 5Division of Hematology and Oncology, Indiana University School of Medicine, 6Department of Molecular Physiology and Biophysics, Vanderbilt University

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

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Medicine

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes
Mark C, Wagner 1, Ruben M. Sandoval 1, Silvia B. Campos-Bilderback 1, Bruce A. Molitoris 1
1Department of Medicine, Division of Nephrology, Indiana University School of Medicine

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

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Developmental Biology

In Ovo Intravascular Injection in Chicken Embryos
Kai Jin 1,2, Jing Zhou 1,2, Gaoyuan Wu 1,2, Ziyi Lian 1,2, Zongyi Zhao 1,2, Shujian Zhou 1,2, Chen Chen 1,2, Hongyan Sun 1,2, Yingjie Niu 1,2, Qishenng Zuo 1,2, Yani Zhang 1,2, Jiuzhou Song 3, Guohong Chen 1,2, Bichun Li 1,2,4
1Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, 2Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, 3Animal & Avian Sciences, University of Maryland, College Park, 4College of Biotechnology, Jiangsu University of Science and Technology

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

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Behavior

The Motivation for Alcohol Reward: Predictors of Progressive-Ratio Intravenous Alcohol Self-Administration in Humans
Bethany L. Stangl 1, Nia D. Byrd 1, Soundarya Soundararajan 1, Martin H. Plawecki 2, Sean O’Connor 2,3, Vijay A. Ramchandani 1
1Human Psychopharmacology Laboratory, Division of Intramural Clinical and Biological Research, NIAAA, 2Dept. of Psychiatry, Indiana University School of Medicine, 3R.L. Roudebush VA Medical Center

This study aims to show that the Progressive-Ratio Computer-assisted Alcohol-Infusion System (CAIS) paradigm is a reliable and sensitive method that can be used to examine the motivating properties associated with alcohol self-administration in humans.

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Biology

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue
Jazmine-Saskya N. Joseph-Chowdhury 1, Stephanie Stransky 1, Sarah Graff 1, Ronald Cutler 1, Dejauwne Young 1, Julie S. Kim 1, Carlos Madrid-Aliste 1, Jennifer T. Aguilan 1, Edward Nieves 1, Yan Sun 1, Edwin J. Yoo 1, Simone Sidoli 1
1Department of Biochemistry, Albert Einstein College of Medicine

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

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Biology

A Modified Technique for Transverse Aortic Constriction in Mice
Kudusi Abuduwufuer *1,2, James Jiqi Wang *1,2, Huihui Li *1,2, Chen Chen 1,2
1Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Hubei Key Laboratory of Genetics and Molecular Mechanism of Cardiological Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

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Medicine

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit
Julian Dilley *1,2,3, Hessam Noori-Dokht *3,4,6, Abhijit Seetharam 1, Margaret Bello 1,3, Aaron Nanavaty 1,3, Roman M. Natoli 2,3, Todd McKinley 1,2,3, Zachary Bault 5, Diane Wagner 2,3,6, Uma Sankar 1,3
1Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, 2Department of Orthopaedic Surgery, Indiana University School of Medicine, 3Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, 4School of Mechanical Engineering, Purdue University, 5Large Animal Resource Center, Indiana University School of Medicine, 6Department of Mechanical and Energy Engineering, Indiana University-Purdue University Indianapolis

The open medial femoral condyle impact model in rabbits is reliable for studying post-traumatic osteoarthritis (PTOA) and novel therapeutic strategies to mitigate PTOA progression. This protocol generates an isolated cartilage defect of the posterior medial femoral condyle in rabbits using a carriage-based drop tower with an impactor head.

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Genetics

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting
Kyungchan Kim 1, Solaema Taleb 1, Jisun So 1, Jamie Wann 1, Hyun Cheol Roh 1
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

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Immunology and Infection

Swine Model of Biofilm Infection and Invisible Wounds
Mohamed El Masry 1, Pramod Bhasme 1, Shomita S. Mathew-Steiner 1, Jessica Smith 1, Thomas Smeenge 1, Sashwati Roy 1, Chandan K. Sen 1
1Indiana Center for Regenerative Medicine & Engineering, Indiana University Health Comprehensive Wound Center, Department of Surgery, Indiana University School of Medicine

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

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Bioengineering

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model
Alexei Christodoulides 1, Abigail R. Hall 1, Anushri Umesh 1,2, Nathan J. Alves 1,2
1Department of Emergency Medicine, Indiana University School of Medicine, 2Weldon School of Biomedical Engineering, Purdue University

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

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Biology

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors
Laura C. Brown 1, Jay Chopra 2, Rachel E. Horness 3, Julia C. van Kessel 4
1Department of Chemistry, Indiana University, 2Indiana University School of Medicine, 3Department of Chemistry, Carleton College, 4Department of Biology, Indiana University

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

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Neuroscience

Optimizing Mouse Urodynamic Techniques for Improved Accuracy
Aytak Khabbaz 1,2, Kristen Lynn Cohen 1,2, Shengqi Zhang 1,2,3, Samhita Chakraborty 1,2, Yihong Zhang 1,2, Lingxiao Deng 1,2
1Department of Neurological Surgery, Indiana University School of Medicine, 2Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery, Goodman and Campbell Brain and Spine, Indiana University School of Medicine, 3Department of Rehabilitation Medicine, Zhongda Hospital Southeast University

This protocol provides a guide for waterproofing the skin with cyanoacrylate to prevent urine absorption by fur and skin. It includes instructions for applying the glue to the skin, implanting a bladder catheter, and electrodes for cystometry and external urethral sphincter electromyography recordings in awake mice.

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