In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.
Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.
Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
1Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, 2Department of Biology, North Carolina Central University, Durham, 3Department of Physiology & Pharmacology and Hypertension & Vascular Research Center, Wake Forest University School of Medicine
An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.
Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.
Meatoplasty, surgical management of meatal stenosis.
1JoVE Content Production, 2Department of Ophthalmology, Massachusetts Eye and Ear
Historically, JoVE, The Journal of Visualized Experiments, has focused primarily on biomedical research and has developed subsections for Bioengineering, Clinical and Translational Medicine, Immunology and Infection, and Neuroscience. This July, JoVE launches its Applied Physics section, which includes a range of content from Plasma Physics to Materials Science. We begin the new section with a notable article from Purdue University, where researchers in the Center for Laser-Based Manufacturing are studying.
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.
Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.
Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs
Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.
We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.
This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.
We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.
Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy
1Raymond and Beverly Sackler Foundation, 2The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 3School of Natural Sciences, Institute for Advanced Study, Princeton, New Jersey
We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.
Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
1The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Johns Hopkins University School of Medicine, 3Department of Dermatology, The Johns Hopkins University School of Medicine, 4Department of Surgery, Johns Hopkins University School of Medicine, 5The Sol Goldman Pancreatic Cancer Center, The Johns Hopkins University School of Medicine, 6Yale Cancer Center, Yale School of Medicine, 7The Skip Viragh Center for Pancreatic Cancer, The Johns Hopkins University School of Medicine, 8Department of Pathology, The Johns Hopkins University School of Medicine
B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor’s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.
Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease
Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.
Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
1Department of Pediatrics and the Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 2VITACYTE, LLC, 3Department of Medicine, Indiana University School of Medicine, 4Department of Cellular and Integrative Physiology, Indiana University School of Medicine
A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.
Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time
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.
RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.
To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.
Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.
1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, 2Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, 3Department of Biology, Duke University
A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.
1Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Department of Neurological Surgery, University of California, San Francisco, 3Department of Neurology, University of California, San Francisco
In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.
This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.
A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.
1Department of poverty related diseases, Barcelona Centre for International Health Research, 2Confocal Microscopy Unit, University of Barcelona- Scientific and Technological Centers, 3Institució Catalana de Recerca i Estudis Avançats (ICREA)
We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.
This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.
We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.
Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.
Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models
Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison
A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.
Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.
The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.
1Michigan Research Community, Undergraduate Research Opportunity Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan
We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.
In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.
Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.
1Department of Internal Medicine D, Experimental Nephrology, University of Münster, 2Department of Nuclear Medicine, University of Münster, 3European Institute for Molecular Imaging, University of Münster
We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.
Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats
A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.
The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.
Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.
A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.
Here we show how to do retro-orbital injection in adult zebrafish.
We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.
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.
Haptic/Graphic Rehabilitation: Integrating a Robot into a Virtual Environment Library and Applying it to Stroke Therapy
Recently, a vast amount of prospects have come available for human-robot interactive systems. In this paper we outline the integration of a new robotic device with open source software that can rapidly make possible a library of interactive functionality. We then outline a clinical application for a neurorehabilitation application.
1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA
The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.