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Pubmed Article
Effectiveness of GnRH antagonist in the management of subfertile couples undergoing controlled ovarian stimulation and intrauterine insemination: a meta-analysis.
PUBLISHED: 01-01-2014
Recent studies have indicated the use of gonadotropin-releasing hormone antagonists (GnRH-ant) as an adjuvant treatment to prevent premature luteinization (PL) and improve the clinical outcomes in patients undergoing controlled ovarian stimulation (COS) with intrauterine insemination (IUI). However, the results of these studies are conflicting.
Authors: Amanda R. Duselis, Paul B. Vrana.
Published: 04-28-2007
Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female’s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated. Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.
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Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish
Authors: Yali Zhao, Nancy L. Wayne.
Institutions: University of California, Los Angeles .
Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.
Neuroscience, Issue 74, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Physiology, Neuroendocrinology, Neurophysiology, Electrophysiology, Comparative, action potential, gonadotropin-releasing hormone, neuron, brain, teleost, animal model
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Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction
Authors: Ryan Hodges, Masayuki Endo, Andre La Gerche, Elisenda Eixarch, Philip DeKoninck, Vessilina Ferferieva, Jan D'hooge, Euan M. Wallace, Jan Deprest.
Institutions: University Hospitals Leuven, Monash University, Victoria, Australia, Katholieke Universiteit Leuven, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER).
Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.
Medicine, Issue 76, Developmental Biology, Biomedical Engineering, Molecular Biology, Anatomy, Physiology, Cardiology, Fetal Therapies, Obstetric Surgical Procedures, Fetal Development, Surgical Procedures, Operative, intrauterine growth restriction, fetal echocardiography, Doppler ultrasound, fetal hemodynamics, animal model, clinical techniques
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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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Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases
Authors: Dongshan Yang, Jifeng Zhang, Jie Xu, Tianqing Zhu, Yanbo Fan, Jianglin Fan, Y. Eugene Chen.
Institutions: University of Michigan Medical Center, University of Yamanashi.
Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.
Medicine, Issue 81, Apolipoprotein C-III, rabbits, knockout, zinc finger nuclease, cardiovascular diseases, lipid metabolism, ApoCIII
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Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Authors: Raffaele Coppini, Cecila Ferrantini, Alessandro Aiazzi, Luca Mazzoni, Laura Sartiani, Alessandro Mugelli, Corrado Poggesi, Elisabetta Cerbai.
Institutions: University of Florence, University of Florence.
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.
Medicine, Issue 86, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
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Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Authors: Khadija Elhabazi, Safia Ayachi, Brigitte Ilien, Frédéric Simonin.
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
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The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Authors: Sara Tremblay, Vincent Beaulé, Sébastien Proulx, Louis-Philippe Lafleur, Julien Doyon, Małgorzata Marjańska, Hugo Théoret.
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
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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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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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Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)
Authors: Mauricio F. Villamar, Magdalena Sarah Volz, Marom Bikson, Abhishek Datta, Alexandre F. DaSilva, Felipe Fregni.
Institutions: Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, Pontifical Catholic University of Ecuador, Charité University Medicine Berlin, The City College of The City University of New York, University of Michigan.
High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes. Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets.
Medicine, Issue 77, Neurobiology, Neuroscience, Physiology, Anatomy, Biomedical Engineering, Biophysics, Neurophysiology, Nervous System Diseases, Diagnosis, Therapeutics, Anesthesia and Analgesia, Investigative Techniques, Equipment and Supplies, Mental Disorders, Transcranial direct current stimulation, tDCS, High-definition transcranial direct current stimulation, HD-tDCS, Electrical brain stimulation, Transcranial electrical stimulation (tES), Noninvasive Brain Stimulation, Neuromodulation, non-invasive, brain, stimulation, clinical techniques
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Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity
Authors: Sheng Li.
Institutions: University of Texas Health Science Center at Houston , TIRR Memorial Hermann Hospital, TIRR Memorial Hermann Hospital.
Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.
Medicine, Issue 71, Neuroscience, Neurobiology, Anatomy, Physiology, Behavior, electrical stimulation, BreEStim, electrode, voluntary breathing, respiration, inspiration, pain, neuropathic pain, pain management, spasticity, stroke, spinal cord injury, brain, central nervous system, CNS, clinical, electromyogram, neuromuscular electrical stimulation
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Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis
Authors: Christine Beeton, Adriana Garcia, K. George Chandy.
Institutions: University of California, Irvine (UCI).
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.
Immunology, Issue 5, Autoimmune Disease, Animal Model, EAE, Experimental Allergic Encephalomyelitis, Multiple Sclerosis, Immunology, Clinical Scoring, Disease Model, Inflammation, Central Nervous System
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Induction of Experimental Autoimmune Hypophysitis in SJL Mice
Authors: Melissa A. Landek-Salgado, Shey-Cherng Tzou, Hiroaki Kimura, Patrizio Caturegli.
Institutions: The Johns Hopkins University.
Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.
Immunology, Issue 46, autoimmunity, hypophysitis, immunization, SJL mice, Freund's adjuvant
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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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Thermal Ablation for the Treatment of Abdominal Tumors
Authors: Christopher L. Brace, J. Louis Hinshaw, Meghan G. Lubner.
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison.
Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 °C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide. During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13. Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 °C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16. Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.
Medicine, Issue 49, Thermal ablation, interventional oncology, image-guided therapy, radiology, cancer
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Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale
Authors: Barry M. Lester, Lynne Andreozzi-Fontaine, Edward Tronick, Rosemarie Bigsby.
Institutions: Brown University, Women & Infants Hospital of Rhode Island, University of Massachusetts, Boston.
There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.
Behavior, Issue 90, NICU Network Neurobehavioral Scale, NNNS, High risk infant, Assessment, Evaluation, Prediction, Long term outcome
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Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Authors: David A. Goss, Richard L. Hoffman, Brian C. Clark.
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
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Protocol for Long Duration Whole Body Hyperthermia in Mice
Authors: Vikas Duhan, Neha Joshi, P. Nagarajan, Pramod Upadhyay.
Institutions: National Institute of Immunology, National Institute of Immunology.
Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.
Medicine, Issue 66, Anatomy, Physiology, Mouse, Fever, Whole Body Hyperthermia, Temperature Spikes, core body temperature
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Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification
Authors: Ashleigh C. McLean, Nicolas Valenzuela, Stephen Fai, Steffany A.L. Bennett.
Institutions: Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, University of Ottawa , University of Ottawa , Azrieli School of Architecture and Urbanism.
A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.
Medicine, Issue 67, Biochemistry, Immunology, Microbiology, Physiology, Anatomy, estrous cycle, vaginal cytology, hormonal status, murine reproduction, 17-beta-estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, prolactin
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Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence
Authors: Natalia J. Sumi, Eydis Lima, John Pizzonia, Sean P. Orton, Vinicius Craveiro, Wonduk Joo, Jennie C. Holmberg, Marta Gurrea, Yang Yang-Hartwich, Ayesha Alvero, Gil Mor.
Institutions: Yale University School of Medicine, NatureMost Laboratories, Bruker Preclinical Imaging.
Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.
Cancer Biology, Issue 93, ovarian cancer, recurrence, in vivo imaging, tumor burden, cancer stem cells, chemotherapy
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Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices
Authors: Peter J. Hemond, Kelly J. Suter.
Institutions: University of Texas San Antonio - UTSA.
Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3). Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons. The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.
Jove Neuroscience, Issue 36, electrophysiology, simultaneous recording, cell-attached recording, current clamp, brain slice
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