Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.
18 Related JoVE Articles!
Quantitative Autonomic Testing
Institutions: University of Massachusetts Medical School.
Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.
Medicine, Issue 53, Deep breathing, Valsalva maneuver, tilt test, sudomotor testing, Composite Autonomic Severity Score, CASS
Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response
Institutions: Midwestern University.
Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.
Behavior, Issue 93, Rat, Rattus norvegicus, voluntary diving, diving response, diving reflex, autonomic reflex, central integration
Deep Brain Stimulation with Simultaneous fMRI in Rodents
Institutions: University of North Carolina, University of North Carolina, University of North Carolina, University of North Carolina, University of North Carolina.
In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal. Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.
Neuroscience, Issue 84, Electric Stimulation Therapy, Animal Experimentation, Immobilization, Intubation, Models, Animal, Neuroimaging, Functional Neuroimaging, Stereotaxic Techniques, Functional magnetic resonance imaging (fMRI), deep brain stimulation (DBS), blood oxygen level dependent (BOLD), subthalamic nucleus, rodent
Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test
Institutions: Universidade Federal do Rio de Janeiro, Brasil.
Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3
, where it has been recently characterized4
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 μL of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5
. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.
Neuroscience, Issue 35, neurodegenerative disease, mice, cell therapy, model
Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1β using a Whole Blood Stimulation Assay
Institutions: National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Arthritis and Musculoskeletal and Skin Diseases.
Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1β 1, 2, 3
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1β upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7
. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1β to its mature, bioactive form 4, 5, 6, 8, 9, 10
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2
. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.
Immunology, Issue 49, Interleukin-1 beta, autoinflammatory, whole blood stimulation, lipopolysaccharide, ATP, cytokine production, pattern-recognition receptors, pathogen-associated molecular patterns
Electrophysiological Recording in the Brain of Intact Adult Zebrafish
Institutions: University of Georgia, University of Georgia, Oklahoma State University, University of Georgia, University of California, Davis.
Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo
electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.
Neuroscience, Issue 81, Zebrafish, adult, Electrophysiology, in vivo, craniotomy, perfusion, neural activity
A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery
Institutions: The Ohio State University.
Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.
Medicine, Issue 86, Myocardial Ischemia/Reperfusion, permanent ligation, left anterior descending artery, myocardial infarction, LAD, ligation, Cardiac troponin I
A Murine Model of Subarachnoid Hemorrhage
Institutions: University of Munich Medical Center.
In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.
Medicine, Issue 81, Nervous System Diseases, Subarachnoid hemorrhage (SAH), mouse model, filament perforation, intracranial pressure monitoring, blood distribution, surgical technique
Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network
Institutions: Beth Israel Deaconess Medical Center.
The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3
It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6
and autism spectrum disorders 7
whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8
). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.
Neuroscience, Issue 46, Transcranial Magnetic Stimulation, rTMS, fMRI, Default Mode Network, functional connectivity, resting state
The FlyBar: Administering Alcohol to Flies
Institutions: Florida State University, University of Houston.
Fruit flies (Drosophila melanogaster
) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g.
, CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.
Neuroscience, Issue 87, neuroscience, alcohol sensitivity, Drosophila, Circadian, sedation, biological rhythms, undergraduate research
Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance
Institutions: Simon Fraser University .
Orthostatic tolerance (OT) refers to the ability to maintain cardiovascular stability when upright, against the hydrostatic effects of gravity, and hence to maintain cerebral perfusion and prevent syncope (fainting). Various techniques are available to assess OT and the effects of gravitational stress upon the circulation, typically by reproducing a presyncopal event (near-fainting episode) in a controlled laboratory environment. The time and/or degree of stress required to provoke this response provides the measure of OT. Any technique used to determine OT should: enable distinction between patients with orthostatic intolerance (of various causes) and asymptomatic control subjects; be highly reproducible, enabling evaluation of therapeutic interventions; avoid invasive procedures, which are known to impair OT1
In the late 1980s head-upright tilt testing was first utilized for diagnosing syncope2
. Since then it has been used to assess OT in patients with syncope of unknown cause, as well as in healthy subjects to study postural cardiovascular reflexes2-6
. Tilting protocols comprise three categories: passive tilt; passive tilt accompanied by pharmacological provocation; and passive tilt with combined lower body negative pressure (LBNP). However, the effects of tilt testing (and other orthostatic stress testing modalities) are often poorly reproducible, with low sensitivity and specificity to diagnose orthostatic intolerance7
Typically, a passive tilt includes 20-60 min of orthostatic stress continued until the onset of presyncope in patients2-6
. However, the main drawback of this procedure is its inability to invoke presyncope in all individuals undergoing the test, and corresponding low sensitivity8,9
. Thus, different methods were explored to increase the orthostatic stress and improve sensitivity.
Pharmacological provocation has been used to increase the orthostatic challenge, for example using isoprenaline4,7,10,11
or sublingual nitrate12,13
. However, the main drawback of these approaches are increases in sensitivity at the cost of unacceptable decreases in specificity10,14
, with a high positive response rate immediately after administration15
. Furthermore, invasive procedures associated with some pharmacological provocations greatly increase the false positive rate1
Another approach is to combine passive tilt testing with LBNP, providing a stronger orthostatic stress without invasive procedures or drug side-effects, using the technique pioneered by Professor Roger Hainsworth in the 1990s16-18
. This approach provokes presyncope in almost all subjects (allowing for symptom recognition in patients with syncope), while discriminating between patients with syncope and healthy controls, with a specificity of 92%, sensitivity of 85%, and repeatability of 1.1±0.6 min16,17
. This allows not only diagnosis and pathophysiological assessment19-22
, but also the evaluation of treatments for orthostatic intolerance due to its high repeatability23-30
. For these reasons, we argue this should be the "gold standard" for orthostatic stress testing, and accordingly this will be the method described in this paper.
Medicine, Issue 73, Anatomy, Physiology, Biomedical Engineering, Neurobiology, Kinesiology, Cardiology, tilt test, lower body negative pressure, orthostatic stress, syncope, orthostatic tolerance, fainting, gravitational stress, head upright, stroke, clinical techniques
Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI
Institutions: University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario.
Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.
Neuroscience, Issue 84, Central Nervous System, Ear, Animal Experimentation, Models, Animal, Functional Neuroimaging, Brain Mapping, Nervous System, Sense Organs, auditory cortex, BOLD signal change, hemodynamic response, hearing, acoustic stimuli
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
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
Basic Surgical Techniques in the Göttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion
Institutions: Aarhus University Hospital, Aarhus University, Aarhus University Hospital.
The emergence of the Göttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6
.The size of the Göttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8
. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion.
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6
. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9
. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of Göttingen minipigs are available via the internet at https://www.minipigs.com10
. For extensive information about porcine anatomy we refer to Nickel et al
. Vol. 1-511
Medicine, Issue 52, Animal model, Animal preparation, Animal handling, Sus scrofa, Swine, Tissue fixation
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
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
Roux-en-Y Gastric Bypass Operation in Rats
Institutions: University Hospital Zürich, University of Zürich, University of Zürich, Imperial College London .
Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2
. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3
. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5
, increased energy expenditure6,7
, altered preference for food high in fat and sugar8,9
, altered salt and water handling of the kidney10
as well as alterations in gut microbiota11
. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15
. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15
. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.
Medicine, Issue 64, Physiology, Roux-en-Y Gastric bypass, rat model, gastric pouch size, gut hormones
Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope
Institutions: VA San Diego Healthcare System, University of California, San Diego, University of California, San Diego.
Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.
Medicine, Issue 86, lung physiology, endotracheal intubation, laryngoscopy, airway resistance, intubation technique
Electrophysiological Measurements and Analysis of Nociception in Human Infants
Institutions: University College London, Great Ormond Street Hospital, University College Hospital, University of Oxford.
Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.
Neuroscience, Issue 58, pain, infant, electrophysiology, human development