1Dr. William M. Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Chicago Medical School, Rosalind Franklin University of Medicine and Science
Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.
Published February 28, 2013. Keywords: Medicine, Genetics, Genomics, Physiology, Anatomy, Biomedical Engineering, Metabolomics, Zebrafish, diabetes, metabolic memory, tissue regeneration, streptozocin, epigenetics, Danio rerio, animal model, diabetes mellitus, diabetes, drug discovery, hyperglycemia
1Department of Nutrional Sciences, University of Wisconsin-Madison, 2Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, 3School of Pharmacy, University of Waterloo
Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function.
Published June 25, 2014. Keywords: Physiology, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
1Department of Surgery, University of California, San Francisco, 2Department of Surgery, Veterans Affairs Medical Center, San Francisco, 3VipeRx Lab, Veterans Affairs Medical Center, San Francisco
Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.
Published October 22, 2014. Keywords: Medicine, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
1Department of GI-, Thorax- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 2Molecular Diabetology, Paul Langerhans Institute Dresden, 3Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden
The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.
Published July 30, 2011. Keywords: Medicine, human islets, Diabetes mellitus, partial pancreatectomy, human islet isolation
1Molecular and Cellular Biochemistry, Center for Molecular Neurobiology, The Ohio State University, 2Integrated Biomedical Science Graduate Program, The Ohio State University, 3Comprehensive Cancer Center, The Ohio State University
Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.
Published April 13, 2011. Keywords: Medicine, islet isolation, islet transplantation, diabetes, murine, pancreas
1Division of Nephrology-Hypertension, Department of Medicine, University of California, San Diego, 2San Diego VA Healthcare System
Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.
Published May 10, 2013. Keywords: Medicine, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Nephrology, Kidney Function Tests, Glomerular filtration rate, rats, mice, conscious, creatinine, inulin, Jaffe, hypertension, HPLC, animal model
JoVE Immunology and Infection
1Department of Microbiology & Immunology, Pennsylvania State University College of Medicine
We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.
Published May 6, 2013. Keywords: Immunology, Medicine, Cellular Biology, Molecular Biology, Microbiology, Anatomy, Physiology, Biomedical Engineering, Genetics, Surgery, Type 1 diabetes, CD4+ T cells, diabetogenic T cells, T cell transfer, diabetes induction method, diabetes, T cells, isolation, cell sorting, FACS, transgenic mice, animal model
1The Heart Research Institute, 2Sydney Medical School, University of Sydney, 3Cardiology Department, Royal Prince Alfred Hospital
A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.
Published May 28, 2013. Keywords: Medicine, Anatomy, Physiology, Biomedical Engineering, Surgery, Tissue, Lacerations, Soft Tissue Injuries, Wound Infection, Wounds, Nonpenetrating, Penetrating, Growth Substances, Angiogenesis Modulating Agents, Wounds and Injuries, Wound healing, mouse, angiogenesis, diabetes mellitus, splint, surgical techniques, animal model
1Umeå Centre for Molecular Medicine, Umeå University, 2Cell Transplant Center, Diabetes Research Institute, University of Miami,, 3EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, 4Dept. of Computing Science, Umeå University
We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.
Published January 12, 2013. Keywords: Medicine, Biomedical Engineering, Cellular Biology, Molecular Biology, Biophysics, Pancreas, Islets of Langerhans, Diabetes Mellitus, Imaging, Three-Dimensional, Optical Projection Tomography, Beta-cell Mass, Near Infrared, Computational Processing
1Department of Chemical and Biological Engineering, Princeton University
We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (http://www.proteinwisdom.org). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.
Published July 25, 2013. Keywords: Genetics, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
1Department of Surgery, University of Louisville
We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.
Published May 2, 2012. Keywords: Medicine, Wound, ischemia, rabbit, minimally invasive, model, diabetes, physiology
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary
The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.
Published February 7, 2011. Keywords: Medicine, Metabolism, Diabetes, Insulin Sensitivity, Methodology
1Department of Pediatrics, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine
This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.
Published June 25, 2012. Keywords: Medicine, Physiology, beta cell, gene expression, islet, diabetes, insulin secretion, proliferation, adenovirus, rat
1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University
We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.
Published March 3, 2013. Keywords: Biomedical Engineering, Medicine, Anatomy, Physiology, Surgery, Immunology, Hematology, Microvessels, Capillaries, Arterioles, Venules, Vascular Diseases, Ischemia, spinotrapezius, peripheral vascular disease, functional vasodilation, arteriolar ligation, vessels, circulation, confocal microscopy, animal model
1Molecular Diabetology, Paul Langerhans Institute Dresden, 2Department of GI-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 3Department of Endocrinology and Metabolism, Metabolic Unit University of Pisa, 4Labs DC0522, Lilly Corporate Center, 5Genomics, Faculty of Medicine Imperial College London, 6Vital-IT, SIB Swiss Institute of Bioinformatics, 7Clinical Biochemistry, Hannover Medical School, 8Cell Physiology and Metabolism, Medical School, University of Geneva, 9Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden, 10R&D DIAB Division / Translational Medicine, Sanofi-Aventis
Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.
Published January 6, 2013. Keywords: Medicine, Physiology, Anatomy, Biochemistry, Cellular Biology, Molecular Biology, Immunology, Surgery, Diabetes Mellitus, Type 2, laser microdissection, dissection, human beta cells, intrinsic autofluorescence, pancreas, partial resection, Diabetes type 2, transcriptomic studies, RNA analysis, islet
1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet
A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.
Published March 10, 2013. Keywords: Medicine, Molecular Biology, Biomedical Engineering, Immunology, Ophthalmology, Surgery, Calcium Metabolism Disorders, Glucose Metabolism Disorders, Diabetes Mellitus, Hyperglycemia, Hyperinsulinism, Hypoglycemia, Transplantation, pancreatic islets, islet, intraocular, anterior chamber, eye, cornea, living window, in vivo imaging, immune responses, cannula, imaging, animal model
1Chemical and Physical Biology Program, Vanderbilt University, 2Department of Physical Medicine and Rehabilitation, Vanderbilt University School of Medicine, 3Radiology & Radiological Sciences, Vanderbilt University Medical Center, 4Department of Pharmacology, Vanderbilt University
The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 °C) and cold conditions (17 °C) in order to map brown adipose tissue (BAT) in adult human subjects.
Published February 18, 2015. Keywords: Medicine, magnetic resonance imaging, brown adipose tissue, cold-activation, adult human, fat water imaging, fluorodeoxyglucose, positron emission tomography, computed tomography
1Department of Biomedical Sciences, Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Department of Cell Biology and Anatomy, Rosalind Franklin University of Medicine and Science
Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.
Published April 8, 2014. Keywords: Developmental Biology, Zebrafish, lateral line regeneration, lateral line development, neuromasts, hair cell regeneration, disease models
1Department of Pharmacology and Toxicology, Wright State University, 2Human Movement Laboratory, Universidade São Judas Tadeu
Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.
Published May 17, 2012. Keywords: Neuroscience, Diabetes, painful diabetic neuropathy, nociception, thermal nociceptive threshold, nocifensive behavior
JoVE Immunology and Infection
1Departments of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida
Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.
Published June 16, 2011. Keywords: Immunology, cell, Type 1 Diabetes, Autoimmunity, Cytotoxic T Lymphocyte
1Department of Pharmacology and Physiology, Rutgers New Jersey Medical School, 2Center for Taste and Feeding Behavior, Universite de Bourgogne
The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.
Published November 27, 2013. Keywords: Neuroscience, membrane potential dye, ventromedial hypothalamus, adult neurons, glucose sensing, fluorescence imaging, arcuate nucleus
JoVE Immunology and Infection
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine
Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.
Published May 22, 2013. Keywords: Immunology, Cellular Biology, Molecular Biology, Biophysics, Physiology, Anatomy, Biomedical Engineering, Surgery, Metabolic Diseases, Diabetes Mellitus, diabetes, Endocrine System Diseases, adipose tissue, AT, stromal vascular fraction, macrophage, lymphocyte, T cells, adipocyte, inflammation, obesity, cell, isolation, FACS, flow cytometry, mice, animal model
1Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, 2The Ritchie Centre, Monash Institute of Medical Research, Monash University
We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.
Published December 21, 2014. Keywords: Medicine, Amnion Membrane, Amniotic, Stem Cells, Epithelial, Cell Therapy, Perinatal, Placenta
1Baker IDI Heart and Diabetes Institute
The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.
Published March 6, 2012. Keywords: Medicine, Lipids, lipoproteins, atherosclerosis, trafficking, cholesterol
1Division of Cardiovascular Medicine, University of Pennsylvania, Perelman School of Medicine, 2Department of Radiology, University of Pennsylvania, Perelman School of Medicine, 3Department of Dermatology, University of Pennsylvania, Perelman School of Medicine
There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.
Published May 2, 2012. Keywords: Medicine, FDG-PET/CT, atherosclerosis, vascular inflammation, quantitative radiology, imaging
1Metabolic Research Unit & Molecular & Medical Research SRC, Geelong, Australia, School of Medicine, Deakin University
Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.
Published January 14, 2013. Keywords: Developmental Biology, Genetics, Biochemistry, Cellular Biology, Molecular Biology, Physiology, Embryology, Metabolism, Metabolomics, metabolic profile, respiration, mitochondria, ATP, development, Oil Red O staining, zebrafish, Danio rerio, animal model
1Department of Mechanical Engineering, University of Michigan-Dearborn, 2Department of Surgery/Transplant, University of Illinois at Chicago, 3Department of Bioengineering, University of Illinois at Chicago
Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.
Published November 17, 2013. Keywords: Bioengineering, Islets of Langerhans, Microfluidics, Microfluidic Analytical Techniques, Microfluidic Analytical Techniques, oxygen, islet, hypoxia, intermittent hypoxia
JoVE Immunology and Infection
1Department of Biology, Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University, 2Department of Chemistry and Biochemistry, Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University, 3Department of Exercise Science, Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University
Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.
Published December 23, 2014. Keywords: Immunology, Periodic acid Schiff, glycogen, soleus muscle, lymphocyte, peripheral blood mononuclear cells, metabolism, immunology, amylase
1Department of Surgery, University of Illinois, Chicago
Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.
Published May 26, 2009. Keywords: Medicine, Human islets, Type 1 diabetes, pancreatic tissue, digestion, human islet transplantation
1Biomedical Engineering Program, University of South Carolina, 2Chemical Engineering Department, University of South Carolina
A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.
Published December 16, 2011. Keywords: Bioengineering, Cell encapsulation, PEG, cell aggregation, hypoxia, insulin secretion, fluorescent imaging
1Department of Surgery, University of Illinois, Chicago
Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.
Published May 26, 2009. Keywords: Medicine, Human islets, Type 1 diabetes, human islet purification, human islet transplantation
1Division of Infectious Diseases, University of Arizona, 2Arizona Center on Aging, University of Arizona
Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.
Published July 24, 2013. Keywords: Medicine, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
1Department of Medicine, Weill Cornell Medical College, 2Department of Oral Biology, University of Missouri-Kansas City-School of Dentistry, 3Department of Pharmacology and Toxicology, University of Missouri Kansas City- School of Pharmacy, 4Regional Hospital, Bamenda, NWP, Cameroon, 5Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, 6Institute for Human Genetics and Biochemistry
A CD4 enumeration method, the α-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The α-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.
Published May 16, 2012. Keywords: Medicine, CD4 count, saliva, antitrypsin, hematopoiesis, T cells, HIV/AIDS, clinical
1Department of Bioengineering, University of Pittsburgh, 2Department of Chemical Engineering, University of Pittsburgh
The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.
Published March 27, 2012. Keywords: Stem Cell Biology, Human embryonic stem cells, Endothelial cells, Pancreatic differentiation, Co-culture
1Stem Cell Lab, School of Psychiatry, Faculty of Medicine, The University of New South Wales, 2Siriraj Center of Excellence for Stem cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 3Neuropsychiatric Institute, Prince of Wales Hospital
We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.
Published March 9, 2012. Keywords: Bioengineering, Alginate microcapsule, 3D platform, embryonic stem cells, definitive endoderm, dopaminergic neurons
1Department of Cardiovascular Medicine, Stanford University
A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.
Published October 15, 2010. Keywords: Medicine, endothelium, endothelial dysfunction, Endo-PAT 2000, peripheral arterial tone, reactive hyperemia
1Wyoming Medical Center
When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (<40 mg/dL), clinical hypoglycemia (<70 mg/dL), and hyperglycemia (>180 mg/dL) also can be observed.
Published June 11, 2012. Keywords: Medicine, Physiology, Computerized glucose management, Endotool, hypoglycemia, hyperglycemia, diabetes, IV insulin, paper protocol, glucose control