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Northwestern University Feinberg School of Medicine

23 ARTICLES PUBLISHED IN JoVE

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Biology

Methods for the Study of the Zebrafish Maxillary Barbel
Elizabeth E. LeClair 1, Jacek Topczewski 2
1Department of Biological Sciences, DePaul University, 2Children’s Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

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Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models
Laura Serwer 1, Rintaro Hashizume 1, Tomoko Ozawa 1, C. David James 1
1Department of Neurological Surgery, University of California, San Francisco - UCSF

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

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Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons
Deepak P. Srivastava 1, Kevin M. Woolfrey 1, Peter Penzes 1,2
1Department of Physiology, Northwestern University Feinberg School of Medicine, 2Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

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Neuroscience

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development
Ben Yang *1, Lauren B. Geary *1, Yong-Chao Ma 1,2
1Northwestern University Feinberg School of Medicine, Children's Hospital of Chicago Research Center, 2Departments of Pediatrics, Neurology and Physiology, Northwestern University Feinberg School of Medicine

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

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Neuroscience

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo
Marin Manuel 1, C.J. Heckman 1
1Department of Physiology, Northwestern University Feinberg School of Medicine

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

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JoVE Core

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices
Staci E. Engle 1, Hilary J. Broderick 1, Ryan M. Drenan 1
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

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Immunology and Infection

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis
Marsha L Quick 1, Joseph D Done 1, Praveen Thumbikat 1
1Department of Urology, Northwestern University Feinberg School of Medicine

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

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Neuroscience

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model
Jonathan C. Chou 1, Stuart D. Rollins 1, Amani A. Fawzi 1
1Department of Ophthalmology, Northwestern University Feinberg School of Medicine

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

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Biology

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice
Keng Jin Lee 1, Lyubov Czech 1, Gregory B. Waypa 1, Kathryn N. Farrow 1
1Department of Pediatrics, Northwestern University Feinberg School of Medicine

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

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Biology

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
Huilin Huang *1, Yilin Xu *1, Chonghui Cheng 1
1Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

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Developmental Biology

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures
Heather H. Ross 1, Milap S. Sandhu 1, Sharareh Sharififar 1, David D. Fuller 1
1Department of Physical Therapy, University of Florida

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level “priming effect” on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

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Bioengineering

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development
Joseph S. Uzarski 1,2, Jimmy Su 1,2,3,4, Yan Xie 1,2, Zheng J. Zhang 1,2, Heather H. Ward 5, Angela Wandinger-Ness 6, William M. Miller 7,8, Jason A. Wertheim 1,2,3,4,8,9
1Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, Feinberg School of Medicine, Northwestern University, 3Department of Biomedical Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, 5Department of Internal Medicine, University of New Mexico HSC, 6Department of Pathology, University of New Mexico HSC, 7Department of Chemical and Biological Engineering, Northwestern University, 8Chemistry of Life Processes Institute, Northwestern University, 9Department of Surgery, Jesse Brown VA Medical Center

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

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Medicine

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma
Aaron J. Clark 1, Shayan Fakurnejad 2, Quanhong Ma 2, Rintaro Hashizume 2,3
1Department of Neurological Surgery, University of California San Francisco, 2Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, 3Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

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Medicine

A Mouse Model for Laser-induced Choroidal Neovascularization
Ronil S. Shah 1, Brian T. Soetikno 1, Michelle Lajko 1, Amani A. Fawzi 1
1Department of Ophthalmology, Northwestern University Feinberg School of Medicine

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

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Bioengineering

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics
Adrian Buganza Tepole 1, Elbert E. Vaca 2, Chad A. Purnell 2, Michael Gart 2, Jennifer McGrath 2, Ellen Kuhl 3, Arun K. Gosain 2
1Mechanical Engineering, Purdue University, 2Division of Plastic Surgery, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, 3Mechanical Engineering, Bioengineering, Cardiothoracic Surgery, Stanford University

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

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Biochemistry

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation
Pan Liu 1, Tomokazu Souma 1, Andrew Zu-Sern Wei 1, Xueying Xie 3, Xunrong Luo 2, Jing Jin 1
1Division of Nephrology and Hypertension, and the Center for Kidney Research and Therapeutics at the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, 2Surgery-Organ Transplantation, Northwestern University Feinberg School of Medicine, 3School of Biological Sciences and Medical Engineering, Southeast University

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

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Cancer Research

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer
Jonathan F. Anker 1, Hanlin Mok 1, Anum F. Naseem 1, Praveen Thumbikat *1,3, Sarki A. Abdulkadir *1,2,3
1Department of Urology, Northwestern University Feinberg School of Medicine, 2The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, 3Department of Pathology, Northwestern University Feinberg School of Medicine

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

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Neuroscience

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine
Matthew C. Arvin 1, David L. Wokosin 2, Sambashiva Banala 3, Luke D. Lavis 3, Ryan M. Drenan 1
1Department of Pharmacology, Northwestern University Feinberg School of Medicine, 2Department of Physiology, Northwestern University Feinberg School of Medicine, 3Janelia Research Campus, Howard Hughes Medical Institute

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

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Cancer Research

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors
Alexander P. Glaser 1, Daniele Procissi 2, Yanni Yu 3, Joshua J. Meeks 3
1Department of Surgery, Division of Urology, NorthShore University HealthSystem, 2Department of Radiology, Northwestern University Feinberg School of Medicine, 3Department of Urology, Northwestern University Feinberg School of Medicine

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

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Kidney Organoid Generation at the Air-Liquid Interface
Ashwani Kumar Gupta 1,2, David Z. Ivancic 1, Bilal A. Naved 1,3, Jason A. Wertheim 1,2,3,4,5,6, Leif Oxburgh 7
1Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, 2Department of Surgery, Northwestern University Feinberg School of Medicine, 3Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology, Northwestern University, 5Chemistry of Life Processes Institute, Northwestern University, 6Department of Surgery, Jesse Brown VA Medical Center, 7The Rogosin Institute

This protocol describes asynchronous mixing of human embryonic stem cells derived kidney progenitors at the air-liquid interface to efficiently generate kidney organoids.

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JoVE Core

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length
Amy N. Adkins 1,4,5, Wendy M. Murray 1,2,3,4,5
1Department of Biomedical Engineering, Northwestern University, 2Department of Physical Medicine & Rehabilitation, Northwestern University Feinberg School of Medicine, 3Department of Physical Therapy and Human Movement Sciences, Northwestern University Feinberg School of Medicine, 4Shirley Ryan AbilityLab, 5Edward Hines, Jr. VA Hospital

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.

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Neuroscience

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing
Hongkai Wang 1,2, Dom D’Andrea 1, Yeon Sik Choi 3,4, Yasmine Bouricha 1, Grace Wickerson 3,4, Hak-Young Ahn 3, Hexia Guo 3,4, Yonggang Huang 3,4,5,6, Milap S. Sandhu 7, Sumanas W. Jordan 8, John A. Rogers 3,4,6,9,10,11,12, Colin K. Franz 1,3,13
1Laboratory of Regenerative Rehabilitation, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 2Northwestern University Interdepartmental Neuroscience Program, 3Center for Bio-integrated Electronics, Querrey Simpson Institute for Bioelectronics, Northwestern University, 4Department of Materials Science and Engineering, Northwestern University, 5Department of Civil and Environmental Engineering, Northwestern University, 6Department of Mechanical Engineering, Northwestern University, 7Arms and Hands Lab, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 8Division of Plastic and Reconstructive Surgery, Biologics, Shirley Ryan AbilityLab, Northwestern University, 9Department of Biomedical Engineering, Northwestern University, 10Department of Neurological Surgery, Northwestern University, 11Department of Chemistry, Northwestern University, 12Department of Electrical and Computer Engineering, Northwestern University, 13The Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

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Neuroscience

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain
Arun Upadhyay 1, Robert J. Vassar 1, Jeffrey N. Savas 1
1Department of Neurology, Northwestern University Feinberg School of Medicine

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

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