Diagnosis-Related Groups:
A system for classifying patient care by relating common characteristics such as diagnosis, treatment, and age to an expected consumption of hospital resources and length of stay. Its purpose is to provide a framework for specifying case mix and to reduce hospital costs and reimbursements and it forms the cornerstone of the prospective payment system.
JoVE Neuroscience
DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord
Developmental Neurobiology, Max Delbrück Center for Molecular Medicine
The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.
JoVE Neuroscience
Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine
An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.
JoVE Neuroscience
The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System
Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.
JoVE Neuroscience
Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method
1Department of Neurosurgery, Cedars Sinai Medical Center, UCLA, 2Basic Medicine School, Fourth Military Medical University, 3Department of Neurology, David Geffen School of Medicine, UCLA, 4Aerospace Medicine School, Fourth Military Medical Univeristy
In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.
JoVE General
Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures
1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School
Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.
JoVE Bioengineering
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Biomedical Engineering, Tulane University
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
JoVE Neuroscience
Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones
Department of Neuroscience, University of Minnesota
A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.
JoVE Neuroscience
Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury
1Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen
We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.
JoVE Neuroscience
Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification
1Michigan Research Community, Undergraduate Research Opportunity Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan
We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.
JoVE Neuroscience
Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays
Department of Biological Sciences, Purdue University
We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.
JoVE Neuroscience
A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells
Biozentrum, University of Basel
Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.
JoVE Neuroscience
Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa
This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).
JoVE Neuroscience
Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth
We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.
JoVE Bioengineering
Axon Stretch Growth: The Mechanotransduction of Neuronal Growth
1Departments of Biomedical Engineering, New Jersey Institute of Technology, 2Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey
A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.
JoVE Clinical and Translational Medicine
The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice
1The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, 2Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen
The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.
JoVE Neuroscience
Live Imaging of Dorsal Root Axons after Rhizotomy
1Temple University, Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, 2Medical Research Service, Department of Veterans Affairs Hospital, 3Department of Neurobiology and Anatomy, Drexel University College of Medicine, 4Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine
An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.
JoVE Neuroscience
Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro
Institute of Reconstructive Neurobiology, University of Bonn
A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.
JoVE Neuroscience
Analyzing Murine Schwann Cell Development Along Growing Axons
1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg
Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.
JoVE Immunology and Infection
Production and Titering of Recombinant Adeno-associated Viral Vectors
1School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, 2Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, 3Department of Biochemistry and Molecular Biophysics, Columbia University
Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.
JoVE Neuroscience
Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons
1Institute for Cellmorphology and molecular Neurobiology, Group for Cellbiology, Ruhr-University Bochum, 2Institute for Clinical Neurobiology, University of Wuerzburg
An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.
JoVE General
Primary Cell Cultures from Drosophila Gastrula Embryos
1Department of Genetics, Harvard Medical School, 2 , Howard Hughes Medical Institute
We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.
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