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Utilization and comparative effectiveness of caspofungin and voriconazole early after market approval in the U.S.
PUBLISHED: 01-01-2014
Both caspofungin and voriconazole were initially approved by the FDA with very narrow indications. Our aim was to evaluate the utilization patterns and comparative effectiveness of these agents early after marketing before any labeling change occurred.
Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd infection with fewer side effects and less treatment-associated mortality.
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Implantation of the Syncardia Total Artificial Heart
Authors: Daniel G. Tang, Keyur B. Shah, Micheal L. Hess, Vigneshwar Kasirajan.
Institutions: Virginia Commonwealth University, Virginia Commonwealth University.
With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of such patients are generally well served by left ventricular assist devices (LVADs). However, a subset of patients with late stage biventricular failure or other significant anatomic lesions are not adequately treated by isolated left ventricular mechanical support. Examples of concomitant cardiac pathology that may be better treated by resection and TAH replacement includes: post infarction ventricular septal defect, aortic root aneurysm / dissection, cardiac allograft failure, massive ventricular thrombus, refractory malignant arrhythmias (independent of filling pressures), hypertrophic / restrictive cardiomyopathy, and complex congenital heart disease. Patients often present with cardiogenic shock and multi system organ dysfunction. Excision of both ventricles and orthotopic replacement with a total artificial heart (TAH) is an effective, albeit extreme, therapy for rapid restoration of blood flow and resuscitation. Perioperative management is focused on end organ resuscitation and physical rehabilitation. In addition to the usual concerns of infection, bleeding, and thromboembolism common to all mechanically supported patients, TAH patients face unique risks with regard to renal failure and anemia. Supplementation of the abrupt decrease in brain natriuretic peptide following ventriculectomy appears to have protective renal effects. Anemia following TAH implantation can be profound and persistent. Nonetheless, the anemia is generally well tolerated and transfusion are limited to avoid HLA sensitization. Until recently, TAH patients were confined as inpatients tethered to a 500 lb pneumatic console driver. Recent introduction of a backpack sized portable driver (currently under clinical trial) has enabled patients to be discharged home and even return to work. Despite the profound presentation of these sick patients, there is a 79-87% success in bridge to transplantation.
Medicine, Issue 89, mechanical circulatory support, total artificial heart, biventricular failure, operative techniques
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Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface
Authors: Qichao Ruan, Janet Moradian-Oldak.
Institutions: University of Southern California.
Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
Bioengineering, Issue 89, Enamel, Amelogenin, Chitosan hydrogel, Apatite, Biomimetic, Erosion, Superficial enamel reconstruction, Dense interface
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A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Authors: Lisa M. Weatherly, Rachel H. Kennedy, Juyoung Shim, Julie A. Gosse.
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here. Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Authors: Lori E. Lowes, Benjamin D. Hedley, Michael Keeney, Alison L. Allan.
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
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Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation
Authors: Amanda K. Rizk, Rima Wardini, Emilie Chan-Thim, Barbara Trutschnigg, Amélie Forget, Véronique Pepin.
Institutions: Concordia University, Concordia University, Hôpital du Sacré-Coeur de Montréal.
Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis. In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.
Medicine, Issue 81, Data tracking, exercise, rehabilitation, adherence, patient compliance, health behavior, user-computer interface.
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Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation
Authors: Ioanna Kosmidou, Shannnon Wooden, Brian Jones, Thomas Deering, Andrew Wickliffe, Dan Dan.
Institutions: Piedmont Heart Institute, Medtronic Inc..
Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion. Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible. We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training. Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.
Medicine, Issue 72, Anatomy, Physiology, Cardiology, Biomedical Engineering, Surgery, Cardiovascular System, Cardiovascular Diseases, Surgical Procedures, Operative, Investigative Techniques, Atrial fibrillation, Cryoballoon Ablation, Pulmonary Vein Occlusion, Pulmonary Vein Isolation, electrophysiology, catheterizatoin, heart, vein, clinical, surgical device, surgical techniques
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Movement Retraining using Real-time Feedback of Performance
Authors: Michael Anthony Hunt.
Institutions: University of British Columbia .
Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes. Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session. This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.
Medicine, Issue 71, Biophysics, Anatomy, Physiology, Physics, Biomedical Engineering, Behavior, Psychology, Kinesiology, Physical Therapy, Musculoskeletal System, Biofeedback, biomechanics, gait, movement, walking, rehabilitation, clinical, training
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Bronchial Thermoplasty: A Novel Therapeutic Approach to Severe Asthma
Authors: David R. Duhamel, Jeff B. Hales.
Institutions: Virginia Hospital Center, 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. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete. Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely. Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.
Medicine, Issue 45, bronchial thermoplasty, severe asthma, airway smooth muscle, bronchoscopy, radiofrequency energy, patient management, moderate sedation
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Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle
Authors: Rosalinda T. Castaneda, Aman Khurana, Ramsha Khan, Heike E. Daldrup-Link.
Institutions: Molecular Imaging Program at Stanford (MIPS) , Stanford University .
Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes. A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo. Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an “off label” use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.
Medicine, Issue 57, USPIO, cell labeling, MR imaging, MRI, molecular imaging, iron oxides, ferumoxytol, cellular imaging, nanoparticles
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The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression
Authors: Jared C. Horvath, John Mathews, Mark A. Demitrack, Alvaro Pascual-Leone.
Institutions: Beth Israel Deaconess Medical Center, Inc..
The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3 In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4 The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053). In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).
Neuroscience, Issue 45, Transcranial Magnetic Stimulation, Depression, Neuronetics, NeuroStar, FDA Approved
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Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans
Authors: Marni J. Falk, Meera Rao, Julian Ostrovsky, Evgueni Daikhin, Ilana Nissim, Marc Yudkoff.
Institutions: The Children's Hospital of Philadelphia, University of Pennsylvania.
Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.
Developmental Biology, Issue 48, Stable isotope, amino acid quantitation, organic acid quantitation, nematodes, metabolism
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Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Authors: Ayman I. Omar.
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1. Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10. There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
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A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Authors: Brian H. Smith, Christina M. Burden.
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides. We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
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Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Authors: F. Aura Kullmann, Stephanie L. Daugherty, William C. de Groat, Lori A. Birder.
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions. The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release. The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
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An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Authors: Justen Manasa, Siva Danaviah, Sureshnee Pillay, Prevashinee Padayachee, Hloniphile Mthiyane, Charity Mkhize, Richard John Lessells, Christopher Seebregts, Tobias F. Rinke de Wit, Johannes Viljoen, David Katzenstein, Tulio De Oliveira.
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
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Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Authors: Kristen K. McCampbell, Kristin N. Springer, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
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Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Authors: Sandra Christoph, Alisa B. Lee-Sherick, Susan Sather, Deborah DeRyckere, Douglas K. Graham.
Institutions: University of Colorado Anschutz Medical Campus, University Hospital of Essen.
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Medicine, Issue 79, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
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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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The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice
Authors: Irving C. Allen.
Institutions: Virginia Polytechnic Institute and State University.
The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.
Infection, Issue 86, LPS, Lipopolysaccharide, mouse, pneumonia, gram negative bacteria, inflammation, acute lung inflammation, innate immunity, host pathogen interaction, lung, respiratory disease
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Using Eye Movements to Evaluate the Cognitive Processes Involved in Text Comprehension
Authors: Gary E. Raney, Spencer J. Campbell, Joanna C. Bovee.
Institutions: University of Illinois at Chicago.
The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Measuring eye movements during reading is one of the most precise methods for measuring moment-by-moment (online) processing demands during text comprehension. Cognitive processing demands are reflected by several aspects of eye movement behavior, such as fixation duration, number of fixations, and number of regressions (returning to prior parts of a text). Important properties of eye tracking equipment that researchers need to consider are described, including how frequently the eye position is measured (sampling rate), accuracy of determining eye position, how much head movement is allowed, and ease of use. Also described are properties of stimuli that influence eye movements that need to be controlled in studies of text comprehension, such as the position, frequency, and length of target words. Procedural recommendations related to preparing the participant, setting up and calibrating the equipment, and running a study are given. Representative results are presented to illustrate how data can be evaluated. Although the methodology is described in terms of reading comprehension, much of the information presented can be applied to any study in which participants read verbal stimuli.
Behavior, Issue 83, Eye movements, Eye tracking, Text comprehension, Reading, Cognition
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The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale
Authors: Ian Q Whishaw, Paul Whishaw, Bogdan Gorny.
Institutions: University of Lethbridge.
Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.
Neuroscience, Issue 18, rat skilled reaching, rat reaching scale, rat, rat movement element rating scale, reaching elements
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Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department
Authors: Franck Verschuren, Maidei Gugu Kabayadondo, Frédéric Thys.
Institutions: Universit Catholique de Louvain Cliniques Universitaires Saint-Luc.
Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.
Medicine, Issue 47, capnography, CO2, emergency medicine, end-tidal CO2
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Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging
Authors: Tobias D. Henning, Sophie Boddington, Heike E. Daldrup-Link.
Institutions: Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco.
In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.
Cell Biology, Issue 13, cell labeling, stem cell, MR imaging, cell tracking, iron oxide, contrast agents, mesenchymal stem cells
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Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects
Authors: Alexander J. Nedopil, Lydia G. Mandrussow, Heike E. Daldrup-Link.
Institutions: Medical Center, University of California San Francisco.
The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2. In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3. Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4. This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.
Cellular Biology, Issue 38, Stem cells, cartilage defect, agarose, scaffold, tissue engineering, implantation, MASI
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Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
Authors: John Marshall, Koji Morikawa, Nicholas Manoukis, Charles Taylor.
Institutions: University of California, Los Angeles.
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
Cellular Biology, Issue 5, mosquito, malaria, popuulation, replacement, modeling, infectious disease
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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.