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Chi tiết tài liệu

Manual of Research Techniques in Cardiovascular Medicine.

Tác giả: Ardehali, Hossein; Bolli, Roberto; Losordo, Douglas W.

Chủ đề:

Chủ đề: Medicine; Cardiovascular; Research Techniques

Nhà xuất bản: John Wiley & Sons

Năm xuất bản: 2014

Loại tài liệu: Book

Mô tả vật lý: 463 p.

Định danh: 978-1-118-49516-2

Ngôn ngữ: en

Đọc trực tuyến

Tải tài liệu

While some research methods or techniques are applicable in several areas of medicine, research in cardiovascular diseases requires knowledge of an increasing array of procedures, techniques and measurements that are highly specialized and unique to this area of investigation. Edited by senior clinical investigators who are recognized leaders in cardiovascular medicine worldwide, this book provides readers with a comprehensive, practical “how-to-do-it” review of best-practice techniques for cardiovascular research.

Cover
Dedication
Title page
Contents
List of Contributors
Preface
About the Companion Website
Part 1: Electrophysiology
- 1: Measurement of calcium transient ex vivo
  - Introduction
  - Preparation and fluorescent indicator loading
  - Optical setup
  - Calibration
  - Analysis and interpretation
  - Set-by-step procedure
  - References
- 2: Confocal imaging of intracellular calcium cycling in isolated cardiac myocytes
  - Introduction
  - Materials and methods
    - Chamber, electrical stimulation, and superfusate delivery apparatus
    - Confocal microscope system
    - Cell loading of fluorescent Ca2+ indicators
    - Data analyses
    - Experimental approach
  - Weaknesses and strengths of the method and alternative approaches
  - Conclusions
  - References
- 3: Generating a large animal model of persistent atrial fibrillation
  - Introduction
    - Insights from tachypacing-induced AF models
  - Protocol
    - Generating a tachypacing-induced AF model
    - Pacing protocols
    - Ventricular rate during AF: to ablate or not to ablate
  - Complications and alternative approaches to study AF
    - Potential complications
    - Alternative AF models
  - Strengths and weaknesses of the model
  - Conclusion
  - Acknowledgement
  - References
- 4: Confocal imaging of intracellular calcium cycling in the intact heart
  - Introduction
  - Measurement of intracellular Ca2+ transients
  - Principles and features of confocal microscopy
  - Procedure
    - Experimental setup
    - Materials and equipment
  - Limitations, potential pitfalls and alternative approaches
  - Additional application: use of confocal imaging to determine efficacy of stem cell integration into host myocardium
  - References
- 5: Recording and measurement of action potentials
  - Introduction
  - Protocol
    - Theory of AP recording
  - Procedure
    - Recording amplifier
    - Choices to make before an experiment
    - AP recording procedure
    - Frequent problems
    - AP analysis
  - Alternative approaches and extensions
  - Conclusion
  - References
  - Weaknesses and strengths
- 6: Patch-clamp recordings from isolated cardiac myocytes
  - Introduction
  - Theory of patch-clamp recording
    - Cell-attached configuration
    - Whole-cell recording configuration
    - Inside-out excised patch configuration
    - Outside-out excised patch configuration
  - Instrumentation for patch-clamp recordings
  - Experimental aspects of patch-clamp recordings
    - Gigaseal formation
    - Whole-cell recordings
    - Whole-cell voltage-clamp protocols, data acquisition, and analysis
  - Conclusions and future directions
  - References
- 7: Optical mapping of the heart
  - Introduction
  - Protocols
    - Experimental set-up
    - Tissue preparations (Figure 7.2)
    - Staining and image acquisition
    - Data processing and analysis
  - Representative results
  - Alternative approaches
  - Weaknesses and strengths of method
  - Conclusions
  - References
Part 2: Isolation and Maintenance of Primary Stem Cells
- 8: Isolation of colony-forming endothelial progenitor cells
  - Introduction
  - Materials
    - Laboratory reagents
    - Laboratory equipment
  - Methods
    - MNC isolation from fresh PB or UCB
    - EPC-CFA of bulk cell populations in PB, GmPB, BM, or UCB
    - Optional-1: EPC-CFA of single CD34+ or CD133+ cells in PB, GmPB, BM, or UCB
    - Optional-2: HELIC assay of single CD34+ or CD133+ cells as cell fate assay of HSC
  - Application
  - Acknowledgements
    - Competing interests statement
  - References
- 9: Cardiac resident stem cells
  - Introduction
  - Protocol
    - Preparation of basic solutions and culture medium
    - Enzymatic digestion of the mouse heart
    - Separation of small cells
    - Sorting c-kit-positive cells using FACS
  - Acknowledgements
  - Alternative approaches
  - Conclusion
  - References
  - Weaknesses and strength of the method
- 10: Cardiospheres
  - Introduction
  - Protocols
    - Preparation for explant creation
    - Explant creation
    - Cardiosphere formation
    - Preparation for cardiosphere formation
  - Alternative approaches
  - Weaknesses and strengths of the method
  - Conclusions
  - References
- 11: Mesenchymal stem cells
  - Introduction
  - Protocols
    - Isolation of human MSCs from BM aspirates
  - Acknowledgements
  - Conclusions
  - References
- 12: Generation and differentiation of human iPS cells
  - Introduction
  - Protocol I: Generation of hiPSC from fibroblasts
    - Production of the VSV-G Lentivirus (6 days)
    - Reprogramming of human fibroblasts (∼14 days)
    - Plasmids and media
    - Titration of the virus
  - Protocol II: differentiation hiPSCs to myocytes
  - Future perspectives
  - References
- 13: Isolation of neonatal and adult rat cardiomyocytes
  - Introduction
  - Protocols
    - Isolation of neonatal rat ventricular cardiomyocytes
    - Isolation of adult rat cardiomyocytes
  - Alternative approaches
  - Weaknesses and strengths of the method
  - Conclusions
  - References
- 14: Isolation and culture of vascular smooth muscle cells
  - Introduction
  - Protocol
    - Reagents
    - Equipment
    - Preparation and equipment setup
    - Procedure
    - Reagent setup
  - Alternative approaches
    - Explant method
  - Conclusion
  - References
  - Strengths and weaknesses of method
- 15: Isolation and culture of cardiac endothelial cells
  - Introduction
  - Protocol for isolation of mouse cardiac endothelial cells
    - Materials and preparations
    - Procedures
    - Important notes
  - Alternative approaches
    - A novel protocol of endothelial cell isolation
  - Conclusions
  - Acknowledgements
  - References
- 16: Isolation and culture of cardiac fibroblasts
  - Introduction
  - Cardiac fibroblasts isolation protocol
    - Materials and preparations
    - Procedures for isolation of mouse cardiac fibroblasts
    - Cell propagation and maintenance
  - Alternative approaches
    - Isolation of cardiac ventricular fibroblasts from rats
  - Conclusions
  - References
- 17: Murine bone marrow transplantation model
  - Introduction
  - Materials
  - Procedure
  - Alternative approaches
  - Animal husbandry
  - Weaknesses and strengths of the method
  - References
- 18: In vitro differentiation and expansion of vascular endothelial cells derived from mouse embryonic stem cells
  - Introduction
  - Materials
    - Tissue culture
  - Methods
    - Differentiation of ES-cells to Flk-1+ progenitors
    - ES-cell culture (see Notes 1 and 2)
    - Purification of Flk-1+ cells
    - Expansion/differentiation of Flk-1+ cells
  - Acknowledgements
  - Notes
  - References
Part 3: Manipulation of the Heart and Vessels in Vivo and ex Vivo
- 19: Coronary ligation
  - Introduction
  - Protocol
  - Alternative approaches
  - Acknowledgments
  - Conclusions
  - References
  - Weaknesses and strengths of the method
- 20: Transverse aortic constriction: a model to study heart failure in small animals
  - Introduction
  - Material
  - Procedure
    - Anesthesia, intubation, and ventilation
    - Preparation of the operative field
    - Aortic banding (constriction of transverse aorta)
    - Notes
    - Postoperative care
  - Limitations
  - Alternative approaches
  - Confirmation of constriction
  - Expected outcomes
  - Weaknesses and strengths of the method
  - Conclusions
  - References
- 21: Pharmacological models of hypertrophy and failure
  - Overview
  - Protocols
  - Alternative approaches
  - Conclusions
  - References
  - Strengths and weaknesses of the method
- 22: Hindlimb ischemia
  - Introduction
  - Protocol
    - Hair removal prior to surgery
    - Induction of anesthesia
    - Surgery preparation
    - Femoral artery ligation
    - Laser Doppler
  - Alternative methods
  - Common considerations
  - Acknowledgements
  - References
- 23: The Langendorff preparation
  - Introduction
  - Protocol
    - Cannulation of the aorta
    - Perfusion solution constituents, thermoregulation, and mode of delivery
    - Recording of left ventricular pressure, ECG, and coronary flow
  - Alternative approaches
  - Weaknesses and strengths of the method
  - Conclusions
  - References
- 24: Myocarditis and other immunological models of cardiac disease
  - Introduction
  - Protocol
    - Induction of EAM
    - Notes
  - Alternative approaches
  - Acknowledgement
  - Conclusions
  - References
  - Strength and weaknesses of the method
- 25: Models of pacing-induced heart failure
  - Introduction
  - Rapid pacing models protocols
  - Functional and hemodynamic expectations
  - Cellular and biochemical considerations to chronic rapid pacing
  - Neurohormonal aspects and expectations
  - Electrophysiology and arrhythmogenesis in chronic rapid pacing
  - Matrix remodeling and chronic rapid pacing
  - Chronic pacing model for therapeutic research and development: technical considerations and limitations
  - Chronic rapid pacing-induced heart failure: the issue of reversibility
  - Acknowledgements
  - Summary
  - References
- 26: Porcine myocardial ischemia models
  - Introduction
  - Methods
    - Anesthetized pig model with an angioplasty balloon-induced occlusion
    - Conscious pig model with an extravascular balloon-induced occlusion
  - Conclusion
  - Acknowledgements
  - References
- 27: Angiogenesis assays
  - In vivo Matrigel plug assay
    - Equipment
    - Matrigel harvest and analysis
    - Matrigel mixtures and injections
    - Preparation of reagents and animals
    - Reagents for quantification of angiogenesis
    - Notes
  - Introduction
  - Mouse corneal micropocket angiogenesis assay
    - Preparation of reagents and animals
    - Equipment
    - Monitoring and quantification of angiogenesis
    - Preparation of controlled slow-release pellet
    - Surgical procedure
  - Weaknesses and strengths
  - Acknowledgement
  - Alternative approaches
  - Discussion and conclusion
  - References
- 28: Immunohistochemical analysis of cardiac tissue
  - Direct and indirect immunofluorescence
    - Protocol 1: labeling of primary antibody
  - Introduction
  - Frozen or paraffin sections
    - Protocol 2: antigen retrieval by microwaving
  - Ah, those colorful images
    - Protocol 3: multicolor labeling of formalin-fixed, paraffin embedded myocardial sections
  - Do your controls
    - Protocol 4: ligand-blocking of an antibody
  - References
- 29: A murine model of cardiac arrest by exsanguination
  - Introduction
  - Materials and methods
    - Materials
    - Methods
  - Discussion
  - Acknowledgments
  - References
Part 4: Small Animal Imaging
- 30: Blood pressure, telemetry, and vascular measurements in the rodent model
  - Introduction
  - Measurement of blood pressure
    - Noninvasive arterial BP monitoring (indirect methods)
    - Invasive arterial BP monitoring (direct methods)
  - Vascular measurements
    - Doppler (in vivo)
    - In vitro
  - Acknowledgements
  - Developing technologies
  - References
- 31: The setting: imaging conscious, sedated, or anesthetized rodents
  - Preparation of rodent and equipment set-up
  - Effects of anesthesia
  - General steps for preparing mice for imaging studies
  - References
- 32: Echocardiography: standard techniques (M-mode, two-dimensional imaging, and Doppler)
  - Introduction
  - Materials
    - Additional equipment
    - Ultrasound systems
  - Methods
    - Technical considerations
    - Animal preparation and positioning
    - Instrument settings
  - Basic protocol
    - Parasternal long axis (PLAX)
    - Parasternal short axis (PSAX)
    - Apical four-chamber (A4C)
    - Apical five-chamber (A5C)
  - Left ventricular structure and function
    - Chamber dimensions and wall thickness
    - Left ventricular mass
    - Left ventricular systolic function
    - Load-independent assessment of myocardial contractility
    - Left ventricular diastolic function
  - Right ventricular assessment and pulmonary hypertension
  - Color Doppler
  - Assessment of embryonic and neonatal mice
  - Assessment of aortic pathology
  - Conclusions
  - References
- 33: Echocardiography: advanced techniques (tissue Doppler, speckle tracking, and three-dimensional imaging)
  - Introduction
  - Tissue Doppler imaging
    - Basic protocol
    - Instrument settings
    - Principles
    - Applications
    - Limitations
  - Speckle tracking echocardiography
    - Principles
    - Applications
    - Basic protocol
    - Instrument settings
    - Limitations
  - Three-dimensional imaging
    - Principles
    - 3D reconstruction from 2D imaging
    - Acquisition
    - Applications
    - Limitations
    - Volumetric 3D imaging
  - References
- 34: In vivo tomographic cardiac imaging: positron emission tomography and magnetic resonance imaging
  - Introduction
  - Protocols
    - Anesthesia of animals
    - MRI
    - PET
  - Acknowledgements
  - Alternative approaches
  - Conclusion
  - Weaknesses and strength of the methods
  - References
- 35: In vivo hemodynamics
  - Introduction
  - Protocol
    - Additional notes
  - Alternative approaches
  - Strengths/ weaknesses of the method
  - Acknowledgments
  - Conclusions
  - References
Part 5: Metabolism, Mitochondria, and Cell Death
- 36: Fractionation of cardiomyocytes and isolation of mitochondria
  - Introduction
  - Protocols
    - Isolation of mitochondria from whole hearts
    - Isolation of mitochondria from cultured cardiomyocytes
  - Alternative approaches
  - Strengths and weaknesses of the method
  - Conclusions
  - References
- 37: Assessment of glucose and fatty acid metabolism ex vivo
  - Introduction
  - Principle
  - Methods
    - Working heart apparatus
    - Radioactively labeled energy substrates
    - Solutions
    - Experimental protocol
    - Measuring myocardial oxygen consumption (MVO2)
  - Applications of this approach
  - Conclusions
  - Limitations and alternative approaches
  - References
- 38: Quantification and characterization of atherosclerotic lesions in mice
  - Introduction
  - Protocols
    - Pathological screening of mice
    - Atherosclerotic lesion analyses
    - Platelet activation and atherosclerosis
    - Macrophages and atherosclerosis
    - Characterization of lesion composition and nature
    - Lesional apoptosis
  - Alternative approaches
  - Weaknesses and strength of the methods
    - Lesions in atherosclerotic mice
    - Procedural problems
  - Conclusions
  - References
- 39: Assessment of cell death in the heart
  - Death pathways
    - Apoptosis
    - Programmed necrosis
    - Caveats for the assessment of cell death in experimental models
  - Introduction
  - Apoptosis
    - Caspase activation
    - Cytochrome c release
    - TUNEL
    - Complementary approaches
  - Necrosis
    - Entry of propidium iodide into the cell
    - Release of intracellular proteins
    - Loss of inner mitochondrial membrane potential (ΔΨm)
    - Assessing necrosis in vivo
  - Acknowledgements
  - Conclusions
  - References
- 40: Assessment of mitochondrial function in isolated cells
  - Introduction
  - Protocols
    - Respiration rates
    - Cellular ATP
    - Mitochondrial membrane potential
    - Mitochondrial mass and biogenesis
    - Additional measures of mitochondrial function
  - Acknowledgements
  - Alternative approaches
  - Conclusions
  - References
  - Weaknesses and strengths of the method
- 41: Multinuclear NMR spectroscopy of myocardial energetics and substrate utilization in isolated perfused mouse hearts
  - Introduction
  - Protocol
    - Creation of fatty acid stock with 13C stable isotopes
    - Langendorff heart perfusions combined with 31P NMR spectroscopy
    - Analysis of NMR spectra
    - Heart tissue processing and 13C NMR spectroscopy of tissue extracts
  - Alternative approaches
  - Variations of the protocol
  - Conclusions
  - References
  - Strengths and weaknesses of method
- 42: Measurement of reactive oxygen species in cardiovascular disease
  - Introduction
  - Measurement of the superoxide radical (O2)
    - Electro paramagnetic resonance spectroscopy (EPR)
    - Chemiluminescence
    - Fluorescence imaging
    - Measurement of superoxide radical in mitochondria
  - Hydrogen peroxide (H2O2) assays
    - 2′7′-dichlorofluorescein diacetate (DCFH-DA)
    - HyPer: [H2O2]
    - Measurement of hydrogen peroxide in mitochondria
  - Bio-markers
    - DNA damage
    - Lipid peroxidation
    - Thiol-disulfide interchange
  - Measurement of mitochondrial ROS in living cells
  - Acknowledgement
  - Conclusion
  - References
- 43: Assessing autophagy
  - Biological significance of autophagy in the heart
  - Introduction
  - Western blot analysis
  - Microscopy
  - Alternative approaches
  - Interpretation
  - Strengths and weaknesses
  - Conclusions
  - References
- 44: Assessment of cardiomyocyte size
  - Introduction
  - Protocol
    - Collagenase isolation of myocytes
    - Myocyte morphometry
  - Alternative approaches
  - Weaknesses and strength of the method
  - Conclusion
  - Helpful advice
  - References
Part 6: Manipulation of Gene Expression in Vitro and in Vivo
- 45: Generation of Cre-loxP mouse models for conditional knockout and overexpression of genes in various heart cells
  - Introduction
  - Protocol
    - Conditional knockout (cKO) gene targeting
    - Conditional knockin (cKI) gene targeting
  - Strengths and weaknesses
  - Alternative approaches
  - Conclusion
  - References
- 46: Modulation of myocardial genes via use of adenoviral vectors and RNA interference approaches
  - Introduction
  - Protocols
    - Considerations for RNAi transfer
    - Guidelines for siRNA design
    - Guidelines of shRNA design
    - Construction of plasmid vector-based siRNA
    - Determination of siRNA knockdown effects on gene expression in cells
    - Construction of adenoviral vector-based siRNA
    - Transfer of adenoviral vector-based shRNA into the heart
  - Alternative approaches
  - Weaknesses and strengths of the method
  - Acknowledgement
  - Conclusions
  - References
- 47: Overexpression and downregulation of proteins in vitro
  - Introduction
  - Protocols
    - Lipofection
    - Nucleofection
    - Adenoviral transduction
    - Lentiviral transduction
  - Weaknesses and strengths of the method
  - Conclusions
  - References
- 48: In vivo microRNA studies
  - Introduction
  - Protocols
    - Protocol I: Northern blot analysis of miRNAs
    - Protocol II: Real-time detection of miRNA levels
    - Protocol III: Preparation of antimiRs for in vivo administration
  - Weaknesses and strengths of the methods
  - Conclusions
  - References
Part 7: Model Systems
- 49: Vascular and cardiac studies in zebrafish
  - Introduction
  - Protocols
    - Morpholino (MO) or mRNA microinjection of zebrafish embryos
    - Whole mount immunofluorescence of zebrafish embryos
    - Zebrafish live embryo cell tracking
  - Alternative approaches
  - Weaknesses and strengths
  - Conclusion
  - References
- 50: Vascular and cardiac studies in Drosophila
  - Introduction
  - Protocols
    - Measurement of in vivo cardiac chamber dimension by optical coherence tomography (OCT)
    - Characterization of heart wall thickness by histology
  - Conclusions
  - References
Index