University of Rochester Medical Center 23 articles published in JoVE Bioengineering Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement Brian C. Wise1,2, Keshia E. Mora1,2, Whasil Lee1,2,3, Mark R. Buckley1,2 1Department of Biomedical Engineering, University of Rochester, 2Department of Orthopaedics, Center for Musculoskeletal Research, University of Rochester Medical Center, 3Department of Pharmacology and Physiology, University of Rochester Medical Center We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement. Cancer Research Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia Christina M. Kaszuba1,2, Benjamin J. Rodems1,3, Sonali Sharma1,3, Edgardo I. Franco1,2, John M. Ashton1,3,4, Laura M. Calvi1,5, Jeevisha Bajaj1,3 1Wilmot Cancer Institute, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester, 3Department of Biomedical Genetics, University of Rochester Medical Center, 4Genomics Research Center, University of Rochester Medical Center, 5Division of Endocrinology and Metabolism, Department of Medicine, University of Rochester Medical Center Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression. Neuroscience Semi-Automated Analysis of Peak Amplitude and Latency for Auditory Brainstem Response Waveforms Using R Daxiang Na1, Patricia M. White2 1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Neuroscience, Ernst J. Del Monte Institute for Neuroscience, University of Rochester Medical Center This article describes the semi-automated measurement of the amplitudes and latencies of the first five peaks and troughs in the auditory brainstem response waveform. An additional routine compiles and annotates the data into a spreadsheet for experimenter analysis. These free computer routines are executed using the open-source statistical package R. Bioengineering Cantilever Bending of Murine Femoral Necks Emma Knapp1, Hani A. Awad1,2 1Department of Orthopedics, Center for Musculoskeletal Research, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment. Biology Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans Maria I. Lazaro-Pena1, Adam B. Cornwell1, Andrew V. Samuelson1 1Department of Biomedical Genetics, University of Rochester Medical Center Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis. Neuroscience The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors Jaimie Girnis1,2, Tarek Agag1, Tobias Nobiling1,3, Vanessa Sweet1,4, Bokkyu Kim1 1Department of Physical Therapy Education, SUNY Upstate Medical University, 2College of Health and Rehabilitation Sciences: Sargent College Center for Neurorehabilitation, Boston University, 3Department of Physical Medicine and Rehabilitation, University of Rochester Medical Center, 4Rehabilitation Today This protocol is intended to investigate the impact of task conditions on movement strategies in chronic stroke survivors. Further, this protocol can be used to examine if a restriction in elbow extension induced by neuromuscular electrical stimulation causes trunk compensation during goal-directed arm reaches in non-disabled adults. Immunology and Infection A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System Stephen Angeloni1, Andrew Cameron2, Nicole D. Pecora2,3, Sherry Dunbar1 1Luminex Corporation, 2Departments of Pathology and Laboratory Medicine, Clinical Microbiology, University of Rochester Medical Center, 3Department of Microbiology and Immunology, University of Rochester Medical Center A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2. Medicine Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits Cameron Bosinski1, Kyle Wagner1, Xiangping Zhou2, Lynn Liu3, David S. Auerbach1 1Department of Pharmacology, Upstate Medical University, 2Department of Neurology, Upstate Medical University, 3Strong Epilepsy Center, Departments of Neurology, Anesthesiology / Perioperative Medicine, & Pediatrics, University of Rochester Medical Center Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death. Neuroscience Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping Bradford Z. Mahon1,2, Jeffrey A. Mead3, Benjamin L Chernoff2, Maxwell H. Sims5, Frank E. Garcea4, Emily Prentiss5, Raouf Belkhir2, Sam J. Haber1, Sarah B. Gannon5, Steve Erickson5, Kelly A. Wright5, Michael Z. Schmidt5, Audrey Paulzak1, Vanessa C. Milano1, David A. Paul1, Kenneth Foxx1, Madalina Tivarus7,8, Jacob W. Nadler6, Jacqueline M Behr1, Susan O. Smith1, Yan Michael Li1, Kevin Walter1, Webster H. Pilcher1 1Department of Neurosurgery, University of Rochester Medical Center, 2Department of Psychology, Carnegie Mellon University, 3Public Relations and Communications, University of Rochester Medical Center, 4MOSS Rehabilitation Research Institute, Cognitive Neuroscience, 5University of Rochester Medical Center, 6Department of Anesthesiology and Perioperative Medicine, University of Rochester Medical Center, 7Department of Imaging Sciences, University of Rochester Medical Center, 8Department of Neuroscience, University of Rochester Medical Center This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients. Neuroscience In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy Amanda M Sweeney*1, Virginia Plá*1, Ting Du1, Guojun Liu1, Qian Sun1, Sisi Peng1, Benjamin A. Plog1, Benjamin T. Kress1, Xiaowei Wang1, Humberto Mestre1, Maiken Nedergaard1,2 1Center for Translational Neuromedicine, University of Rochester Medical Center, 2Center for Translational Neuromedicine, University of Copenhagen Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull. Immunology and Infection Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone Ginés Ávila-Pérez1, Jun-Gyu Park1, Aitor Nogales1, Fernando Almazán2, Luis Martínez-Sobrido1 1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter. Bioengineering Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation Doran S. Mix1, Michael C. Stoner1, Steven W. Day2, Michael S. Richards3 1Division of Vascular Surgery, University of Rochester Medical Center, 2Department of Biomedical Engineering, Rochester Institute of Technology, 3Department of Surgery, University of Rochester Medical Center Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments. Genetics Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations Alec Kittredge1, Changyi Ji1, Yu Zhang 1, Tingting Yang1 1Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells. Biochemistry Expression and Purification of Mammalian Bestrophin Ion Channels Alec Kittredge1, Nancy Ward1, Austin Hopiavuori1, Yu Zhang 1, Tingting Yang1 1Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels. Genetics The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan Adam B. Cornwell1, Jesse R. Llop1, Peter Salzman2,3, Juilee Thakar4, Andrew V. Samuelson1 1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Biostatistics and Computational Biology, University of Rochester Medical Center, 3Non-Clinical Statistics, Bristol-Myers Squibb, 4Department of Microbiology and Immunology, University of Rochester Medical Center Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data. Neuroscience Cannula Implantation into the Cisterna Magna of Rodents Anna L.R. Xavier1, Natalie Linea Hauglund1, Stephanie von Holstein-Rathlou1, Qianliang Li1, Simon Sanggaard1,2, Nanhong Lou3, Iben Lundgaard3,4, Maiken Nedergaard1,3 1Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Copenhagen, 2Department of Anesthesiology, Yale School of Medicine, 3Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Rochester Medical Center, 4Department of Experimental Medical Science, Wallenberg Center for Molecular Medicine, Lund University Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds. Medicine Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation Jomy J. Varghese1, Isaac L. Schmale2, Mollie Eva Hansen1, Shawn D. Newlands2, Danielle S.W. Benoit1, Catherine E. Ovitt3 1Department of Biomedical Engineering, University of Rochester, 2Department of Otolaryngology, University of Rochester Medical Center, 3Center for Oral Biology, University of Rochester Medical Center We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands. Bioengineering Retroductal Nanoparticle Injection to the Murine Submandibular Gland Jomy J. Varghese1, Isaac L. Schmale2, Yuchen Wang1, Mollie Eva Hansen1, Shawn D. Newlands2, Catherine E. Ovitt3, Danielle S. W. Benoit1 1Department of Biomedical Engineering, University of Rochester, 2Department of Otolaryngology Head and Neck Surgery, University of Rochester Medical Center, 3Center for Oral Biology, University of Rochester Medical Center Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles. Immunology and Infection Influenza A Virus Studies in a Mouse Model of Infection Laura Rodriguez1, Aitor Nogales1, Luis Martínez-Sobrido1 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection. Medicine Murine Flexor Tendon Injury and Repair Surgery Jessica E. Ackerman1, Alayna E. Loiselle1 1Center for Musculoskeletal Research, Department of Orthopaedics & Rehabilitation, University of Rochester Medical Center Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing. Developmental Biology Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration Michael A. Trembley1,3, Lissette S. Velasquez1, Eric M. Small1,2,3 1Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 2Department of Medicine, University of Rochester School of Medicine and Dentistry, 3Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility. Neuroscience Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation Christopher Nishimura1, Oksana Polesskaya2, Stephen Dewhurst1, Jharon N. Silva1 1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Human Genetics, University of Chicago This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy. Biology An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans Bruno B. Queliconi1, Alicia J. Kowaltowski1, Keith Nehrke2 1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 2Department of Medicine, Nephrology Division, University of Rochester Medical Center, School of Medicine and Dentistry A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.
