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.
16 Related JoVE Articles!
Murine Model of Allergen Induced Asthma
Institutions: Emory University and Atlanta VA Medical Center.
Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1
More than 8% of the US population has asthma, with the prevalence increasing.2
As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3
Availability of a variety of transgenic strains further increases the attractiveness of these models.4
Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6
and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7
techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.
Immunology, Issue 63, Allergy, airway hyperresponsiveness, pulmonary function, eosinophil, ovalbumin, methacholine, airway resistance, plethysmography, flexiVent, bronchoalveolar lavage, physiology
Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique
Institutions: McGill University , SCIREQ Scientific Respiratory Equipment Inc..
The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent
; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in naïve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.
Medicine, Issue 75, Biomedical Engineering, Anatomy, Physiology, Biophysics, Pathology, lung diseases, asthma, respiratory function tests, respiratory system, forced oscillation technique, respiratory system mechanics, airway hyperresponsiveness, flexiVent, lung physiology, lung, oxidative stress, ventilator, cannula, mice, animal model, clinical techniques
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
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
Cortical Source Analysis of High-Density EEG Recordings in Children
Institutions: UCL Institute of Child Health, University College London.
EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1
. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2
, because the composition and spatial configuration of head tissues changes dramatically over development3
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.
Behavior, Issue 88, EEG, electroencephalogram, development, source analysis, pediatric, minimum-norm estimation, cognitive neuroscience, event-related potentials
Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
Institutions: University of Pennsylvania , University of Pennsylvania .
parasites are blood flukes that infect an estimated 200 million people worldwide 1
. In chronic infection with Schistosoma
, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2
. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni
eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation.
Following intraperitoneal sensitization and intravenous challenge, S. mansoni
eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6
. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni
eggs from infected livers (modified from 7
), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis.
Overall, this method provides an in vivo
model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.
Immunology, Issue 64, Infection, Microbiology, helminth, parasite, mouse, Th2, lung, inflammation, granuloma, alternative activation, macrophage
Angiogenesis in the Ischemic Rat Lung
Institutions: Johns Hopkins University.
The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1
, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.
Medicine, Issue 72, Anatomy, Physiology, Biomedical Engineering, Pathology, Surgery, Lung, Lung Diseases, Lung Injury, Thoracic Surgical Procedures, Physiological Processes, Growth and Development, Respiratory System, Physiological Phenomena, angiogenesis, bronchial artery, blood vessels, arteries, rat, ischemia, intubation, artery ligation, thoracotomy, cannulation, animal model
Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung
Institutions: National Institutes of Health.
The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.
Medicine, Issue 81, mouse, rodents, intratracheal intubation, delivery of exogenous substances, lung, study of airway pathology and physiology, pulmonary fibrosis
Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
Institutions: McMaster University, Hamilton, University of Toronto.
Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2
. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4
. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5
. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7
. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.
Immunology, Issue 65, Medicine, Physiology, Dendritic Cells, allergic airway inflammation, ovalbumin, lymph nodes, lungs, dendritic cell maturation, dendritic cell migration, mediastinal lymph nodes
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections
Institutions: Universidad de la República, Trinity College Dublin.
Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages.
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae
inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.
Medicine, Issue 90, Sublingual immunotherapy, Pneumonia, Streptococcus pneumoniae, Lungs, Flagellin, TLR5, NLRC4
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
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
Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI
Institutions: King Abdulaziz University, The University of Sheffield.
The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ
assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1
. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3
. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1
of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5
. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3
Immunology, Issue 93, Allergy, Immunology, IgE, Fcε, RI, horse (Equus caballus), Immunoassay
A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice
Institutions: Baylor College of Medicine (BCM), Millenium Premier Group, Baylor College of Medicine (BCM).
Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible. Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples. Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated. Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated. AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined. Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed. These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge. This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.
Physiology, Issue 38, Airway resistance, intubation, airway hyperreactivity, acetylcholine
In vitro Measurements of Tracheal Constriction Using Mice
Institutions: UT Health Science Center, San Antonio.
Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2
. In vitro
tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro
tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1
). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o
and Gq coupled receptors, respectively3,4,5
. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7
signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10
. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11
. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1
) and calcium influx (so called "excitation-contraction coupling")4,7
. More detailed reviews on signaling pathways controlling airway constriction can be found4,12
. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14
, both well-known ASM modulators in humans and other species5,15
Here we present protocols for the isolation of murine tracheal rings and the in vitro
measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+
, Figure 1
). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1
), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1
). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.
Medicine, Issue 64, Physiology, trachea, force transduction, Airway smooth muscle, constriction, cholinergic receptor
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina