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The PKD inhibitor CID755673 enhances cardiac function in diabetic db/db mice.
PUBLISHED: 03-24-2015
The development of diabetic cardiomyopathy is a key contributor to heart failure and mortality in obesity and type 2 diabetes (T2D). Current therapeutic interventions for T2D have limited impact on the development of diabetic cardiomyopathy. Clearly, new therapies are urgently needed. A potential therapeutic target is protein kinase D (PKD), which is activated by metabolic insults and implicated in the regulation of cardiac metabolism, contractility and hypertrophy. We therefore hypothesised that PKD inhibition would enhance cardiac function in T2D mice. We first validated the obese and T2D db/db mouse as a model of early stage diabetic cardiomyopathy, which was characterised by both diastolic and systolic dysfunction, without overt alterations in left ventricular morphology. These functional characteristics were also associated with increased PKD2 phosphorylation in the fed state and a gene expression signature characteristic of PKD activation. Acute administration of the PKD inhibitor CID755673 to normal mice reduced both PKD1 and 2 phosphorylation in a time and dose-dependent manner. Chronic CID755673 administration to T2D db/db mice for two weeks reduced expression of the gene expression signature of PKD activation, enhanced indices of both diastolic and systolic left ventricular function and was associated with reduced heart weight. These alterations in cardiac function were independent of changes in glucose homeostasis, insulin action and body composition. These findings suggest that PKD inhibition could be an effective strategy to enhance heart function in obese and diabetic patients and provide an impetus for further mechanistic investigations into the role of PKD in diabetic cardiomyopathy.
Authors: Raffaele Coppini, Cecila Ferrantini, Alessandro Aiazzi, Luca Mazzoni, Laura Sartiani, Alessandro Mugelli, Corrado Poggesi, Elisabetta Cerbai.
Published: 04-21-2014
Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models. Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method. The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.
22 Related JoVE Articles!
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Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring
Authors: Maria V. Irazabal, Prasanna K. Mishra, Vicente E. Torres, Slobodan I. Macura.
Institutions: Mayo Clinic, Mayo Clinic.
Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.
Medicine, Issue 100, Magnetic Resonance Imaging (MRI), Ultra-high field (UHF) MRI, rodent, phenotype, kidney, cysts, polycystic kidney disease (PKD), Autosomal dominant polycystic kidney disease (ADPKD), Autosomal-recessive polycystic kidney disease (ARPKD), progression, interventions,Total Kidney Volume (TKV).
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Transthoracic Echocardiography in Mice
Authors: Jonathan L. Respress, Xander H.T. Wehrens.
Institutions: Baylor College of Medicine (BCM), Baylor College of Medicine (BCM).
In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.
Medicine, Issue 39, Echocardiography, pulse wave Doppler, tissue Doppler imaging, ultrasound
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A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Authors: Shahram Jevin Poureetezadi, Eric K. Donahue, Rebecca A. Wingert.
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
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Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure
Authors: Ilayaraja Muthuramu, Marleen Lox, Frank Jacobs, Bart De Geest.
Institutions: Catholic University of Leuven.
Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure. Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling. Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.
Medicine, Issue 94, Myocardial infarction, cardiac remodelling, infarct expansion, heart failure, cardiac function, invasive hemodynamic measurements
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A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique
Authors: Lorissa Lamoureux, Jeejabai Radhakrishnan, Raúl J. Gazmuri.
Institutions: Rosalind Franklin University of Medicine and Science.
A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes – but is not limited to – hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.
Medicine, Issue 98, Cardiopulmonary resuscitation, Hemodynamics, Myocardial ischemia, Rats, Reperfusion, Ventilation, Ventricular fibrillation, Ventricular function, Translational medical research
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Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound
Authors: Janet M. Denbeigh, Brian A. Nixon, Mira C. Puri, F. Stuart Foster.
Institutions: University of Toronto, Sunnybrook Research Institute, Mount Sinai Hospital, Toronto.
Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.
Developmental Biology, Issue 97, Micro-ultrasound, Molecular imaging, Mouse embryo, Microbubble, Ultrasound contrast agent, Perfusion
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Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice
Authors: Wei-Ting Chang, Sudeshna Fisch, Michael Chen, Yiling Qiu, Susan Cheng, Ronglih Liao.
Institutions: Brigham and Women's Hospital, Harvard Medical School, Chi-Mei Medical Center, Tainan.
Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.
Medicine, Issue 98, Coronary flow reserve, Doppler echocardiography, non-invasive methodology, use of animals in research, pressure overload, aortic banding
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Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect
Authors: Bo Zhang, Jonathan P. Davis, Mark T. Ziolo.
Institutions: The Ohio State University, Huazhong University of Science and Technology.
Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., β-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.
Medicine, Issue 100, murine, catheterization, contractility, PV loops, end-systolic pressure volume relationship, preload recruitable stroke work, NOS1
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Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets
Authors: Oanh H. Do, Jiun T. Low, Peter Thorn.
Institutions: The University of Queensland.
Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.
Medicine, Issue 99, Pancreatic islets, Leprdb, db/db, isolation, liberase, collagenase, 2-photon imaging, exocytosis, insulin, beta cell, diabetes
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How to Ignite an Atmospheric Pressure Microwave Plasma Torch without Any Additional Igniters
Authors: Martina Leins, Sandra Gaiser, Andreas Schulz, Matthias Walker, Uwe Schumacher, Thomas Hirth.
Institutions: University of Stuttgart.
This movie shows how an atmospheric pressure plasma torch can be ignited by microwave power with no additional igniters. After ignition of the plasma, a stable and continuous operation of the plasma is possible and the plasma torch can be used for many different applications. On one hand, the hot (3,600 K gas temperature) plasma can be used for chemical processes and on the other hand the cold afterglow (temperatures down to almost RT) can be applied for surface processes. For example chemical syntheses are interesting volume processes. Here the microwave plasma torch can be used for the decomposition of waste gases which are harmful and contribute to the global warming but are needed as etching gases in growing industry sectors like the semiconductor branch. Another application is the dissociation of CO2. Surplus electrical energy from renewable energy sources can be used to dissociate CO2 to CO and O2. The CO can be further processed to gaseous or liquid higher hydrocarbons thereby providing chemical storage of the energy, synthetic fuels or platform chemicals for the chemical industry. Applications of the afterglow of the plasma torch are the treatment of surfaces to increase the adhesion of lacquer, glue or paint, and the sterilization or decontamination of different kind of surfaces. The movie will explain how to ignite the plasma solely by microwave power without any additional igniters, e.g., electric sparks. The microwave plasma torch is based on a combination of two resonators — a coaxial one which provides the ignition of the plasma and a cylindrical one which guarantees a continuous and stable operation of the plasma after ignition. The plasma can be operated in a long microwave transparent tube for volume processes or shaped by orifices for surface treatment purposes.
Engineering, Issue 98, atmospheric pressure plasma, microwave plasma, plasma ignition, resonator structure, coaxial resonator, cylindrical resonator, plasma torch, stable plasma operation, continuous plasma operation, high speed camera
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
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Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Authors: Eva Wagner, Sören Brandenburg, Tobias Kohl, Stephan E. Lehnart.
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
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Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy
Authors: Stephen C. Kolwicz Jr., Rong Tian.
Institutions: University of Washington School of Medicine.
Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.
Medicine, Issue 42, cardiac physiology, high energy phosphate, phosphocreatine, ATP
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Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Authors: Bridget Valsamis, Susanne Schmid.
Institutions: University of Western Ontario.
The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3. We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used. Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.
Neuroscience, Issue 55, Startle responses, rat, mouse, sensory gating, sensory filtering, short-term habituation, long-term habituation, prepulse inhibition
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Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice
Authors: Sebastián Dante Calligaris, Micaela Ricca, Paulette Conget.
Institutions: Clínica Alemana Universidad del Desarrollo.
Dobutamine is a β-adrenergic agonist with an affinity higher for receptor expressed in the heart (β1) than for receptors expressed in the arteries (β2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former. Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine. As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.
Medicine, Issue 72, Anatomy, Physiology, Cardiology, Surgery, Cardiovascular System, Cardiovascular Diseases, Life Sciences (General), Computer Programming and Software, cardiac stress test, dobutamine, cardiac catheterization, hemodynamic parameters, mice, animal model
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Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR
Authors: Lik Chuan Lee, Zhang Zhihong, Andrew Hinson, Julius M. Guccione.
Institutions: UCSF/VA Medical Center, LoneStar Heart, Inc..
Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function. Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients. Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces. Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.
Medicine, Issue 74, Biomedical Engineering, Anatomy, Physiology, Biophysics, Molecular Biology, Surgery, Cardiology, Cardiovascular Diseases, bioinjection, ventricular wall stress, mathematical model, heart failure, cardiac function, myocardium, left ventricle, LV, MRI, imaging, clinical techniques
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Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes
Authors: Evan Lee Graham, Cristina Balla, Hannabeth Franchino, Yonathan Melman, Federica del Monte, Saumya Das.
Institutions: Beth Israel Deaconess Medical Center, Harvard Medical School, Sapienza University.
The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.
Cellular Biology, Issue 79, Medicine, Cardiology, Cellular Biology, Anatomy, Physiology, Mice, Ion Channels, Primary Cell Culture, Cardiac Electrophysiology, adult mouse cardiomyocytes, cell isolation, IonOptix, Cell Culture, adenoviral transfection, patch clamp, fluorescent nanosensor
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Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model
Authors: Jonathan C. Chou, Stuart D. Rollins, Amani A. Fawzi.
Institutions: Northwestern University Feinberg School of Medicine.
Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.
Neurobiology, Issue 76, Neuroscience, Biomedical Engineering, Medicine, Anatomy, Physiology, Cellular Biology, Molecular Biology, Ophthalmology, Eye, Posterior Eye Segment, Retinal Diseases, Eye Enucleation, trypsin digest, mouse, rat, rodent, retina, vasculature, blood vessel, histology, diabetes, tissue, animal model
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Echocardiographic Assessment of the Right Heart in Mice
Authors: Evan Brittain, Niki L. Penner, James West, Anna Hemnes.
Institutions: Vanderbilt University Medical Center, Vanderbilt University Medical Center.
Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking. In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.
Medicine, Issue 81, Anatomy, Physiology, Biomedical Engineering, Cardiology, Cardiac Imaging Techniques, Echocardiography, Echocardiography, Doppler, Cardiovascular Physiological Processes, Cardiovascular System, Cardiovascular Diseases, Echocardiography, right ventricle, right ventricular function, pulmonary hypertension, Pulmonary Arterial Hypertension, transgenic models, hemodynamics, animal model
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A Practical Guide to Phylogenetics for Nonexperts
Authors: Damien O'Halloran.
Institutions: The George Washington University.
Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts. We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.
Basic Protocol, Issue 84, phylogenetics, multiple sequence alignments, phylogenetic tree, BLAST executables, basic local alignment search tool, Bayesian models
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Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model
Authors: Ajith Kumar GS, Binil Raj, Santhosh Kumar S, Sanjay G, Chandrasekharan Cheranellore Kartha.
Institutions: Rajiv Gandhi Centre for Biotechnology, Rajiv Gandhi Centre for Biotechnology, Sree Chitra Tirunal Institute for Medical Sciences & Technology.
Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.
Medicine, Issue 88, ascending aorta, cardiac hypertrophy, pressure overload, aortic constriction, thoracotomy, surgical model.
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Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System
Authors: Frans Vinberg, Vladimir Kefalov.
Institutions: Washington University in St. Louis.
An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.
Neuroscience, Issue 99, Electrophysiology, electroretinogram, ERG, ex vivo ERG, retina, photoreceptor, rod, cone, a-wave, b-wave, drug testing
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.