Use of Human Perivascular Stem Cells for Bone Regeneration
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh
Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.
Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia
A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.
Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.
Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.
Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model
1Department of Biomedical Engineering, Duke University, 2School of Medicine, Duke University, 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania
A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.
Murine Model of Hindlimb Ischemia
1Division of Cardiovascular Medicine, Stanford University, 2Department of Anesthesiology, University of California, San Francisco
The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.
Orthotopic Hind-Limb Transplantation in Rats
1Department of Visceral, Transplant, and Thoracic Surgery, Daniel Swarovski Research Laboratory, Innsbruck Medical University, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Pittsburgh Medical Center
Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.
Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements
Department of Pharmacology and Toxicology, Michigan State University
Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.
Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound
1Department of Medical Physics in Radiology, German Cancer Research Center, Heidelberg, Germany, 2Unit of Chemotherapy and Toxicology, German Cancer Research Center, Heidelberg, Germany
In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.
A Swine Model of Neonatal Asphyxia
1Departments of Pediatrics, Pharmacology and Surgery, University of Alberta, 2Department of Surgery, University of Alberta
Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.
Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California
We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.
A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis
1Institute for Molecular Cardiovascular Research, RWTH Aachen University, 2Institute for Textile Technology and Mechanical Engineering, RWTH Aachen University, 3Institute for Applied Medical Engineering, Helmholtz-Institute of RWTH Aachen University, 4Department of Experimental Molecular Imaging, RWTH Aachen University, 5Department of Oral and Maxillofacila Surgery, RWTH Aachen University
A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.
Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures
1Cardiology, Robotic Cardiac Electrophysiology and Arrhythmia Unit, La Paz University Hospital, 2Magnetecs Corp., 3Cardiology, Geffen School of Medicine at UCLA Los Angeles
This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.
Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles
This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.
Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling
1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University
We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.
Basic Surgical Techniques in the Göttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion
1Department of Neurosurgery, Aarhus University Hospital, 2Department of Neurobiology, Institute of Anatomy, Faculty of Health Sciences, Aarhus University, 3Positron Emission Tomography (PET) Centre, Aarhus University Hospital
The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of Göttingen minipigs in biomedical research.
Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure
1Program in Developmental Biology, Children's Memorial Research Center, 2Department of Pediatrics, Northwestern University
In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.
Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock
Department of Surgery, University of Pittsburgh
The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.
The WATCHMAN Left Atrial Appendage Closure Device for Atrial Fibrillation
University of Leipzig Heart Center
The accompanying video describes a procedure for percutaneous placement of the WATCHMAN Left Atrial Appendage (LAA) Device. The WATCHMAN is a nitinol device designed to be permanently implanted at, or slightly distal to, the opening of the left atrial appendage (LAA) to trap blood clots before they exit the LAA, preventing thromboembolic stroke.
The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System
1Department of Radiology, Brain Imaging Center, Academic Medical Center Amsterdam, 2Biological Imaging Centre, MRC Clinical Sciences Centre, Imperial College London
The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.
Gene Transfer for Ischemic Heart Failure in a Preclinical Model
Cardiovascular Research Center, Mount Sinai School of Medicine
A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.
Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Center for Neural Development and Disease, University of Rochester Medical Center, 3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center
Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Department of Medical Microbiology and Immunology, University of Alberta
We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.
Murine Fetal Echocardiography
Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.
Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation
Department of Cardiology, St. Antonius Hospital, The Netherlands
Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.
Single-unit In vivo Recordings from the Optic Chiasm of Rat
Department of Biomedical Engineering, Boston University
Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.
Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels
Department of Neurology, University of Duisburg-Essen Medical School
Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.
Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation
1Department of Cardiac Electrophysiology, Piedmont Heart Institute, 2Cardiac Rhythm, Medtronic Inc.
Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.
Microinjection of Zebrafish Embryos to Analyze Gene Function
1Department of Genetics, Harvard Medical School, 2Department of Cardiology, Children’s Hospital Boston
This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.
Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast
Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University
Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.
Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects
Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.
RNAi Interference by dsRNA Injection into Drosophila Embryos
Department of Biological Sciences, Oakland University
RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.
Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer
1Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, 2Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, 3Department of Surgery, College of Medicine, University of Arizona, Tucson, 4Biomedical Diagnostics and Research, Tucson, AZ, 5Department of Medicine, College of Medicine, University of Arizona, Tucson
Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.
Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects
Department of Orthopaedic Surgery, University of California, San Francisco
This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.
Evaluation of Mammary Gland Development and Function in Mouse Models
Department of Anatomy and Cell Biology, University of Western Ontario
This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.
Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement
Department of Neural and Pain Sciences, University of Maryland
A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.
Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct
Otolaryngology - Head and Neck Surgery, Case Western Reserve University
This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.
Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease
1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine
This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.
Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
1Division of Cardiovascular Medicine, Stanford University, 2Department of Radiology, Stanford University
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.
Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants
Department of Biological Sciences, University of Alabama
This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.
Microvascular Decompression: Salient Surgical Principles and Technical Nuances
1Department of Neurosurgery, Vanderbilt University Medical Center, 2Vanderbilt School of Medicine, Vanderbilt University Medical Center
There are many available options for management of the patient with trigeminal neuralgia. Microvascular decompression, while the most invasive of all options, is also the most effective at achieving long term remission of symptoms. Video instruction on how to maximize efficacy and minimize complications with this procedure is described.
Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine
A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.
A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development
Department of Microbiology and Immunology, Loyola University Medical Center
We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.
Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects
1Department of Neurology, Universitätsmedizin Charité, 2Center for Stroke Research Berlin (CSB), Universitätsmedizin Charité, 3Berlin School of Mind and Brain, Humboldt Universität zu Berlin
Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.
Olfactory Behavioral Testing in the Adult Mouse
1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School
Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.
Zebrafish Brain Ventricle Injection
1Whitehead Institute for Biochemical Research, 2MIT - Massachusetts Institute of Technology
After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.
Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI
We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
Growth Assays to Assess Polyglutamine Toxicity in Yeast
Boston Biomedical Research Institute
This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.
The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease
1German Center for Neurodegenerative Diseases, DZNE, 2Laboratory of Functional Neurogenomics, Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen
Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.
Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice
Department of Biomedical Sciences, Cornell University
In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.
