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Pubmed Article
Jejunal proteins secreted by db/db mice or insulin-resistant humans impair the insulin signaling and determine insulin resistance.
PLoS ONE
PUBLISHED: 01-07-2013
Two recent studies demonstrated that bariatric surgery induced remission of type 2 diabetes very soon after surgery and far too early to be attributed to weight loss. In this study, we sought to explore the mechanism/s of this phenomenon by testing the effects of proteins from the duodenum-jejunum conditioned-medium (CM) of db/db or Swiss mice on glucose uptake in vivo in Swiss mice and in vitro in both Swiss mice soleus and L6 cells. We studied the effect of sera and CM proteins from insulin resistant (IR) and insulin-sensitive subjects on insulin signaling in human myoblasts.
Authors: Curtis C. Hughey, Dustin S. Hittel, Virginia L. Johnsen, Jane Shearer.
Published: 02-07-2011
ABSTRACT
Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.
23 Related JoVE Articles!
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
52010
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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Authors: Joshua C. Neuman, Nathan A. Truchan, Jamie W. Joseph, Michelle E. Kimple.
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Waterloo.
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.
Physiology, Issue 88, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
50374
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Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Authors: Sandra Christoph, Alisa B. Lee-Sherick, Susan Sather, Deborah DeRyckere, Douglas K. Graham.
Institutions: University of Colorado Anschutz Medical Campus, University Hospital of Essen.
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Medicine, Issue 79, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
50720
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Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters
Authors: Ana D. Lopez, Ayse G. Kayali, Alberto Hayek, Charles C. King.
Institutions: University of California, San Diego.
For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.
Medicine, Issue 87, human fetal pancreas, islet cell cluster (ICC), transplantation, immunofluorescence, endocrine cell proliferation, differentiation, C-peptide
50796
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Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice
Authors: Hirotaka Shoji, Keizo Takao, Satoko Hattori, Tsuyoshi Miyakawa.
Institutions: Fujita Health University, Core Research for Evolutionary Science and Technology (CREST), National Institutes of Natural Sciences.
The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.
Behavior, Issue 85, Fear, Learning, Memory, ImageFZ program, Mouse, contextual fear, cued fear
50871
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Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Authors: Raffaele Coppini, Cecila Ferrantini, Alessandro Aiazzi, Luca Mazzoni, Laura Sartiani, Alessandro Mugelli, Corrado Poggesi, Elisabetta Cerbai.
Institutions: University of Florence, University of Florence.
Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models. Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method. The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.
Medicine, Issue 86, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
51116
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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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Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Authors: Jacob Michael Froehlich, Iban Seiliez, Jean-Charles Gabillard, Peggy R. Biga.
Institutions: University of Alabama at Birmingham, INRA UR1067, INRA UR1037.
Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.
Basic Protocol, Issue 86, myogenesis, zebrafish, myoblast, cell culture, giant danio, moustached danio, myotubes, proliferation, differentiation, Danioninae, axolotl
51354
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Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
Authors: Daniella Kovacsics, Jayne Raper.
Institutions: Hunter College, CUNY.
Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).
Genetics, Issue 87, hydrodynamic gene delivery, hydrodynamics-based transfection, mouse, gene therapy, plasmid DNA, transient gene expression, tail vein injection
51481
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Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection
Authors: Abigail Betanzos, Michael Schnoor, Rosario Javier-Reyna, Guillermina García-Rivera, Cecilia Bañuelos, Jonnatan Pais-Morales, Esther Orozco.
Institutions: Center for Research and Advanced Studies of the National Polytechnic Institute, Center for Research and Advanced Studies of the National Polytechnic Institute, Center for Research and Advanced Studies of the National Polytechnic Institute.
Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.
Immunology, Issue 88, Entamoeba histolytica, EhCPADH112, cell adhesion, MDCK, Caco-2, tight junction disruption, amoebiasis, host-pathogen interaction, infection model, actin cytoskeleton
51668
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Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells
Authors: Charles Zhang, Arthur T. Suckow, Steven D. Chessler.
Institutions: University of California, San Diego, Janssen Research & Development, University of California, San Diego.
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function. Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions. Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
Medicine, Issue 76, Cellular Biology, Molecular Biology, Biomedical Engineering, Immunology, Hepatology, Islets of Langerhans, islet, Insulin, Coculture, pancreatic beta cells, INS-1 cells, extracellular contact, transmembrane protein, transcellular interactions, insulin secretion, diabetes, cell culture
50365
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A Model of Chronic Nutrient Infusion in the Rat
Authors: Grace Fergusson, Mélanie Ethier, Bader Zarrouki, Ghislaine Fontés, Vincent Poitout.
Institutions: CRCHUM, University of Montreal.
Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.
Biomedical Engineering, Issue 78, Medicine, Anatomy, Physiology, Basic Protocols, Surgery, Metabolic Diseases, Infusions, Intravenous, Infusion Pumps, Glucolipotoxicity, Rat, Infusion, Glucose, Intralipid, Catheter, canulation, canula, diabetes, animal model
50267
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Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells
Authors: Ulrike Liisberg Aune, Lauren Ruiz, Shingo Kajimura.
Institutions: University of California, San Francisco , University of Copenhagen, Denmark, National Institute of Nutrition and Seafood Research, Bergen, Norway.
Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.
Cellular Biology, Issue 73, Medicine, Anatomy, Physiology, Molecular Biology, Surgery, Adipose Tissue, Adipocytes, Transcription Factors, Cell Differentiation, Obesity, Diabetes, brown adipose tissue, beige/brite cells, primary adipocytes, stromal-vascular fraction, differentiation, uncoupling protein 1, rosiglitazone, differentiation, cells, isolation, fat, animal model
50191
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A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation
Authors: Erik J. Zmuda, Catherine A. Powell, Tsonwin Hai.
Institutions: The Ohio State University, The Ohio State University, The Ohio State University.
Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation. Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.
Medicine, Issue 50, islet isolation, islet transplantation, diabetes, murine, pancreas
2096
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An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice
Authors: J. Andries Ferreira, Jacqueline M. Crissey, Marybeth Brown.
Institutions: University of Missouri, Columbia, University of Missouri, Columbia.
The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls. Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation. We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 ± 2.9g) did not significantly decline immediately after 14d of HU (22.7 ± 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 ± 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p<0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice. The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.
Physiology, Issue 49, Hindlimb unloading, suspension, tail-traction, mice, animal model, atrophy
2467
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Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Authors: Bridget Valsamis, Susanne Schmid.
Institutions: University of Western Ontario.
The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3. We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used. Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.
Neuroscience, Issue 55, Startle responses, rat, mouse, sensory gating, sensory filtering, short-term habituation, long-term habituation, prepulse inhibition
3446
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A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells
Authors: Anne-Sophie Arnold, Martine Christe, Christoph Handschin.
Institutions: University of Basel.
Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures. Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium. The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context. Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.
Neuroscience, Issue 62, Human primary muscle cells, embryonic spinal cord explants, neurites, innervation, contraction, cell culture
3616
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Roux-en-Y Gastric Bypass Operation in Rats
Authors: Marco Bueter, Kathrin Abegg, Florian Seyfried, Thomas A. Lutz, Carel W. le Roux.
Institutions: University Hospital Zürich, University of Zürich, University of Zürich, Imperial College London .
Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12. During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly. Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.
Medicine, Issue 64, Physiology, Roux-en-Y Gastric bypass, rat model, gastric pouch size, gut hormones
3940
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Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice
Authors: Julio E. Ayala, Deanna P. Bracy, Carlo Malabanan, Freyja D. James, Tasneem Ansari, Patrick T. Fueger, Owen P. McGuinness, David H. Wasserman.
Institutions: Sanford-Burnham Medical Research Institute at Lake Nona, Vanderbilt University School of Medicine, Vanderbilt University School of Medicine, Indiana University School of Medicine.
Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd). The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.
Medicine, Issue 57, Glucose, insulin, clamp, mice, insulin resistance, diabetes, liver, muscle, conscious, restraint-free, non-stressed
3188
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Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry
Authors: Shyny Koshy, Parema Alizadeh, Lubov T. Timchenko, Christine Beeton.
Institutions: Baylor College of Medicine.
Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes. The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3. The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies. Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.
Cellular Biology, Issue 45, Glucose, FACS, Plasma Membrane, Insulin Receptor, myoblast, myocyte, adipocyte
2429
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A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device
Authors: Adeola F. Adewola, Yong Wang, Tricia Harvat, David T. Eddington, Dongyoung Lee, Jose Oberholzer.
Institutions: University of Illinois, Chicago, University of Illinois, Chicago.
A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 μm diameter and 150 μm depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 μm in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.
Cellular Biology, Issue 35, Microfluidics, Islet perifusion, glucose ramp, imaging, perifusion, beta cells, insulin secretion
1649
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Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection
Authors: Sang Mo Kang.
Institutions: University of California, San Francisco - UCSF.
Issue 7, Immunology, Heterotopic Heart Transplant, Small Bowel Transplant, Transplant Rejection, T regs, Diabetes, Autoimmune Disease, Translational Research
256
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In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos
Authors: William Walantus, David Castaneda, Laura Elias, Arnold Kriegstein.
Institutions: University of California, San Francisco - UCSF.
In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video.
Neuroscience, Issue 6, Protocol, electroporation, Injection, Stem Cells, brain, transfection
239
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