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Pubmed Article
Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells.
PLoS ONE
PUBLISHED: 01-01-2014
P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis.
ABSTRACT
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
24 Related JoVE Articles!
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An Experimental System to Study Mechanotransduction in Fetal Lung Cells
Authors: Yulian Wang, Zheping Huang, Pritha S. Nayak, Juan Sanchez-Esteban.
Institutions: Alpert Medical School of Brown University.
Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.
Bioengineering, Issue 60, Mechanical stretch, differentiation, lung injury, isolation, fetal, type II epithelial cells, fibroblasts
3543
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Pseudomonas aeruginosa Induced Lung Injury Model
Authors: Varsha Suresh Kumar, Ruxana T. Sadikot, Jeanette E. Purcell, Asrar B. Malik, Yuru Liu.
Institutions: University of Illinois at Chicago, Emory University, University of Illinois at Chicago.
In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.
Immunology, Issue 92, Lung, injury, pseudomonas, pneumonia, mouse model, alveoli
52044
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Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
Authors: Rahul Kushwah, Jim Hu.
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
4014
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In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Authors: Yanning Wu, Shuo Wang, Chunying Li.
Institutions: Wayne State University School of Medicine, Wayne State University School of Medicine, Wayne State University School of Medicine.
Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7. Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18. Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.
Biochemistry, Issue 66, Molecular Biology, Chemistry, CFTR, macromolecular complex, protein interaction, PDZ scaffold protein, epithelial cell, cystic fibrosis
4091
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In vitro Measurements of Tracheal Constriction Using Mice
Authors: Iurii Semenov, Jeremiah T. Herlihy, Robert Brenner.
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. M2/Gi/o 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
3703
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Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy
Authors: Fei Liu, Daniel J. Tschumperlin.
Institutions: Harvard School of Public Health.
Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.
Biophysics, Issue 54, Atomic force microscopy, indentation, stiffness, fibrosis, extracellular matrix
2911
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Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example
Authors: Arava Shatil-Cohen, Hadas Sibony, Xavier Draye, François Chaumont, Nava Moran, Menachem Moshelion.
Institutions: The Hebrew University of Jerusalem, Université catholique de Louvain, Université catholique de Louvain.
Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield Pf.
Plant Biology, Issue 92, Osmotic water permeability coefficient, aquaporins, protoplasts, curve fitting, non-instantaneous osmolarity change, volume change time course
51652
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Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture
Authors: Mark A. LaBarge, James C. Garbe, Martha R. Stampfer.
Institutions: Lawrence Berkeley National Laboratory.
Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries. The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities. Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.
Cancer Biology, Issue 71, Medicine, Anatomy, Physiology, Cellular Biology, Tissue Culture, Tissue Engineering, Oncology, Human mammary epithelial cell culture, reduction mammoplasty, mastectomy, breast cancer, tumor, cancer, matrigel, cell culture
50011
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Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres
Authors: Virginia Espina, Kirsten H. Edmiston, Lance A. Liotta.
Institutions: George Mason University, Virginia Surgery Associates.
Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue. Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.
Cancer Biology, Issue 93, Breast, ductal carcinoma in situ, epidermal growth factor, mammosphere, organoid, pre-invasive, primary cell culture, serum-free, spheroid
51926
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Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation
Authors: Hon Wai Koon, Samantha Ho, Michelle Cheng, Ryan Ichikawa, Charalabos Pothoulakis.
Institutions: The University of California Los Angeles, Los Angeles.
To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.
Immunology, Issue 68, Genetics, Cellular Biology, Physiology, Bone marrow transplantation, colitis, mice, irradiation
4208
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Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
Authors: Harpreet Singh, Sarah Warburton, Thomas M. Vondriska, Baljit S. Khakh.
Institutions: David Geffen School of Medicine, University of California, Los Angeles, David Geffen School of Medicine, University of California, Los Angeles, David Geffen School of Medicine, University of California, Los Angeles.
Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain. P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 & TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.
Neuroscience, Issue 27, Pull down, recombinant protein, GST, brain, rat, mass spectrometry, protein interactions, P2X2, macromolecular complex, channel, receptor, purinergic
1178
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Procedure for Lung Engineering
Authors: Elizabeth A. Calle, Thomas H. Petersen, Laura E. Niklason.
Institutions: Yale University, Duke University, Yale University.
Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.
Bioengineering, Issue 49, Decellularization, tissue engineering, lung engineering, lung tissue, extracellular matrix
2651
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Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung
Authors: Yan Cai, Shioko Kimura.
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
50601
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Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices
Authors: Renate Paddenberg, Petra Mermer, Anna Goldenberg, Wolfgang Kummer.
Institutions: Justus-Liebig-University.
Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 µm in diameter3. We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 µm inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 µm which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 µm which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.
Medicine, Issue 83, Hypoxic pulmonary vasoconstriction, murine lungs, precision cut lung slices, intra-pulmonary, pre- and intra-acinar arteries, videomorphometry
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A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Authors: George Papadopoulos, Carolyn D. Kramer, Connie S. Slocum, Ellen O. Weinberg, Ning Hua, Cynthia V. Gudino, James A. Hamilton, Caroline A. Genco.
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation. Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90, Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
51556
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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Authors: Melissa V. Fernandez, Elizabeth A. Miller, Nina Bhardwaj.
Institutions: New York University School of Medicine, Mount Sinai Medical Center, Mount Sinai Medical Center.
Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5. Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion6. Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin. Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.
Immunology, Issue 87, NLRP3, inflammasome, IL-1beta, Interleukin-1 beta, dendritic, cell, Nigericin, Toll-Like Receptor 8, TLR8, R848, Monocyte Derived Dendritic Cells
51284
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Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies
Authors: Marcus Gereke, Andrea Autengruber, Lothar Gröbe, Andreas Jeron, Dunja Bruder, Sabine Stegemann-Koniszewski.
Institutions: Helmholtz Centre for Infection Research, Otto-von-Guericke University , Helmholtz Centre for Infection Research.
Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended. Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3. In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.
Immunology, Issue 70, Cellular Biology, Molecular Biology, Infection, Infectious Diseases, Microbiology, alveolar type II epithelial cells, mouse, respiratory tract, lung, cell sorting, flow cytometry, influenza, autoimmunity
4322
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
51278
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Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Authors: Alison X. Xie, Kelli Lauderdale, Thomas Murphy, Timothy L. Myers, Todd A. Fiacco.
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
51458
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Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography
Authors: Rebecca Lim, Marcus J. Zavou, Phillipa-Louise Milton, Siow Teng Chan, Jean L. Tan, Hayley Dickinson, Sean V. Murphy, Graham Jenkin, Euan M. Wallace.
Institutions: Monash Institute of Medical Research, Monash Medical Centre, Animal Resource Centre, Perth, Australia, Wake Forest Institute for Regenerative Medicine.
Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.
Physiology, Issue 90, Unrestrained Whole Body Plethysmography, Lung function, Respiratory Disease, Rodents
51755
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The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice
Authors: Irving C. Allen.
Institutions: Virginia Polytechnic Institute and State University.
The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.
Infection, Issue 86, LPS, Lipopolysaccharide, mouse, pneumonia, gram negative bacteria, inflammation, acute lung inflammation, innate immunity, host pathogen interaction, lung, respiratory disease
51391
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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
51807
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Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Authors: Kristen K. McCampbell, Kristin N. Springer, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
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Direct Tracheal Instillation of Solutes into Mouse Lung
Authors: My N. Helms, Edilson Torres-Gonzalez, Preston Goodson, Mauricio Rojas.
Institutions: Emory University, Emory University, Emory University.
Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 μL instillate volume can be introduced into mouse lung.
Medicine, Issue 42, trachea, instillation, distal lung, alveolar space, survival surgery
1941
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