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.
Transverse Aortic Constriction in Mice
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)
Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.
Acute Myocardial Infarction in Rats
1Department of Internal Medicine, Division of Cardiology, University of Texas Medical Branch, 2Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston (UH), Texas Medical Center
The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.
Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations
A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.
Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR
1Department of Surgery, UCSF/VA Medical Center, 2Clinical & Regulatory, LoneStar Heart, Inc.
This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.
Quantitative Analysis of Chromatin Proteomes in Disease
1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah
Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.
Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing
1Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 2Harvard Stem Cell Institute
The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.
Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes
Department of Internal Medicine B, University Medicine Greifswald
The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.
Isolation of Functional Cardiac Immune Cells
Department of Cell Biology and Anatomy, University of South Carolina- School of Medicine
This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.
Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens
1Department of Surgery, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Biology, University of Minnesota, 4Department of Integrative Biology & Physiology, University of Minnesota, 5Institute for Engineering in Medicine, University of Minnesota
The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.
Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury
1Department of Anesthesiology and Perioperative Medicine, Oregon Health & Sciences University, 2Department of Pharmacology, University of Colorado Denver
A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.
Murine Echocardiography and Ultrasound Imaging
1Department of Pharmacology and Physiology, University of Rochester, 2Aab Cardiovascular Research Institute, University of Rochester, 3Visualsonics, 4Department of Medicine, University of Rochester
This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.
A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and Ease of Implantation
Heart Center Rostock, University Hospital of Rostock, Germany
Dual-chamber implantable cardioverter-defibrillators (ICDs) may improve detection of atrial fibrillation as well as differentiation of tachycardias. However, this advantage is undermined by complications associated with the second electrode, which is required in conventional dual chamber devices. Therefore, BIOTRONIK has developed a new electrode called the LinoxSMART S DX that, when used in conjunction with the Lumax DX ICD, offers dual-chamber detection without the risks associated with the second electrode.
A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion
1Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Germany, 2Institute of Physiology, University of Bonn, Germany
Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.
Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction
Department of Physiology, Brody School of Medicine, East Carolina University
Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.
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.
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.
Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster
Department of Geriatric Medicine, University of Michigan Medical School
We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.
Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy
Department of Anesthesiology & Pain Medicine, University of Washington School of Medicine
Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.
Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart
Department of Biomedical Engineering, Washington University in St. Louis
This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.
Quantitative Autonomic Testing
Department of Neurology, University of Massachusetts Medical School
Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.
Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow
Department of Biomedical Engineering, University of Wisconsin – Madison
The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.
Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure
Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University
This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.
Synthesis of an In vivo MRI-detectable Apoptosis Probe
1Division of Cardiovascular Medicine, Department of Medicine, Stanford University Medical Center, 2Division of Cardiology, Department of Medicine, University of California, San Francisco, 3San Francisco VAMC
Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.
Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model
Chagas Disease Multidisciplinary Research Laboratory, University of Brasilia
The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.
Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary
Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.
Mouse Models for Graft Arteriosclerosis
1Department of Surgery, Yale University School of Medicine, 2Department of Pathology, Yale University School of Medicine
We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.
Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance
Department of Biomedical Physiology and Kinesiology, Simon Fraser University
We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined.
Assessing Endothelial Vasodilator Function with the Endo-PAT 2000
Department of Cardiovascular Medicine, Stanford University
A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.
Transthoracic Echocardiography in Mice
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)
Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.
March 2013: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the March 2013 issue of Journal of Visualized Experiments (JoVE).
Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center
We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.
Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine
A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.
Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children
1Department of Public Health, Academic Medical Center - University of Amsterdam, 2Department of Epidemiology, Documentation and Health Promotion, Public Health Service of Amsterdam (GGD), 3Department of Biological Psychology, VU University, 4EMGO+ Institute, VU University Medical Center, 5Institute of Health Sciences, VU University, 6Department of Pediatrics, VU University Medical Center
Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.
Orthotopic Small Bowel Transplantation in Rats
1Department of Surgery, University of Bonn, Germany, 2Department of Surgery, Kyoto University Hospital
Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.
Methods for Intravenous Self Administration in a Mouse Model
Addictions Unit, McGill University Health Centre
The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.
Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice
1Department of Biomedical Engineering, Saint Louis University, 2Department of Internal Medicine, Washington University School of Medicine
Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
1Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST), Eberhard Karls University, Tübingen, 2Department of Cell and Tissue Engineering, Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany, 3Department for Medical Interfacial Engineering (IGVT), University of Stuttgart, Germany, 4Institute of Tissue Engineering and Regenerative Medicine, Julius-Maximillians University, Würzburg, Germany
Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.
Ambulatory ECG Recording in Mice
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)
Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.
Small Bowel Transplantation In Mice
Department of Surgery, University of California, San Francisco - UCSF
The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.
Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult
Department of Biomedical Engineering, Saint Louis University
Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.
Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs
1Program for the Study and Control of Tropical Diseases -PECET-School of Medicine, University of Antioquia, 2School of Agrarian Sciences, University of Antioquia
Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.
Bronchial Thermoplasty: A Novel Therapeutic Approach to Severe Asthma
1Director of Pulmonary Special Procedures and the Lung Cancer Center, Virginia Hospital Center, 2Chief, Division of Pulmonary Critical Care and Sleep Medicine, Virginia Hospital Center
Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.
Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes
1Experimental and Clinical Research Center (ECRC), Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC), 2Medical Department, Division of Cardiology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 3Medical Department, Division of Cardiology and Angiology, Campus Mitte, Charité - Universitätsmedizin Berlin
Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.
Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence
1Department of Medicine, Albert Einstein College of Medicine, 2Diabetes Research and Training Center, Albert Einstein College of Medicine, 3Department of Genetics, Albert Einstein College of Medicine
An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.
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.
In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations
Institute of Molecular Life Sciences, University of Zurich
A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.
Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies
Department of Biology, University of Kentucky, Lexington
We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.
A Novel Ex vivo Culture Method for the Embryonic Mouse Heart
McAllister Heart Institute, University of North Carolina at Chapel Hill
Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.
