The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.
A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.
A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale
Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.
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.
Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat
Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.
The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice
1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida
The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.
Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats
1CullenWebb Animal Neurology & Ophthalmology Center, Riverview, NB, 2Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, 3Department of Comparative Biology and Experimental Medicine, University of Calgary, 4Department of Neuroscience, University of Calgary
Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.
The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.
Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo
We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.
The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.
A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.
This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle’s contractile response at 37 degrees C with an intact circulation.
1Department of Physiology and Pharmacology, Loma Linda University School of Medicine, 2College of Natural and Agricultural Sciences, University of California, Riverside, 3Department of Anesthesiology, Loma Linda University School of Medicine, 4Department of Neurosurgery, Loma Linda University School of Medicine
Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.
Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons
This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.
Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo
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.
Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury
Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.
Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.
Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.
Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.
A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing >95% loss of striatal dopamine and 90.63±-4.02 % ipsiversive rotational bias towards the side of the lesion.
1Department of Neurology, University of Connecticut Health Center, 2Department of Neurology, School of Medicine, University of Pennsylvania, 3Department of Neurosurgery, Hartford Hospital, 4Department of Neurosurgery, School of Medicine, University of Pennsylvania
The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)
1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine
This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.
1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI)
In this video we demonstrate the preparation of E18 Cortical Rat Neurons.
Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
1Institute for Memory Impairments and Neurological Disorders, University of California, 2Physical Medicine & Rehabilitation, University of California, 3Anatomy & Neurobiology, University of California, 4Sue and Bill Gross Stem Cell Research Center, University of California, 5Section of Molecular Biology, University of California, 6Reeve-Irvine Research Center, University of California
Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.
Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.
Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay
A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.
Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat
1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 3Department of Physiology and Functional Genomics, University of Florida, 4Department of Neurology, University of Florida
Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.
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.
Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.
Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall
To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.
Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.
Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs
Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.
1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University
The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.
We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.
The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.
1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine
An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.
Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques
Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.
The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.
Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion
1Department of Physiology and Functional Genomics, University of Florida, 2Department of Neurosurgery, McKnight Brain Institute, University of Florida, 3Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.
1Department of Hematology and Oncology, Brigham and Women's Hospital and Harvard Medical School, 2Department of Cell Biology and Genetics, Erasmus University Medical Center, 3Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital and Harvard Medical School
This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.
Computer-generated stimuli using the Jacky dragon as a model.
Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.
1Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal, 2Division of Experimental Medicine and Program in Neuroengineering, McGill University, 3Program in Neuroengineering, McGill University, 4Montreal Neurological Institute, 5Department of Anatomy and Cell Biology, McGill University, 6Department of Biology, McGill University, 7Department of Medicine, Universite de Montreal - University of Montreal
This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.
Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.
Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.
Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Institute of Diabetes Obesity and Metabolism, Institute for Regenerative Medicine, Department of Pediatrics, University of Pennsylvania-School of Medicine
We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.
DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.
This article describes a method for labeling embryonic skin and thymus blood vessels.