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A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations.
PUBLISHED: 02-28-2011
Portal hypertension (PH) is a common complication and a leading cause of death in patients with chronic liver diseases. PH is underlined by structural and functional derangement of liver sinusoid vessels and its fenestrated endothelium. Because in most clinical settings PH is accompanied by parenchymal injury, it has been difficult to determine the precise role of microvascular perturbations in causing PH. Reasoning that Vascular Endothelial Growth Factor (VEGF) is required to maintain functional integrity of the hepatic microcirculation, we developed a transgenic mouse system for a liver-specific-, reversible VEGF inhibition. The system is based on conditional induction and de-induction of a VEGF decoy receptor that sequesters VEGF and preclude signaling. VEGF blockade results in sinusoidal endothelial cells (SECs) fenestrations closure and in accumulation and transformation of the normally quiescent hepatic stellate cells, i.e. provoking the two processes underlying sinusoidal capillarization. Importantly, sinusoidal capillarization was sufficient to cause PH and its typical sequela, ascites, splenomegaly and venous collateralization without inflicting parenchymal damage or fibrosis. Remarkably, these dramatic phenotypes were fully reversed within few days from lifting-off VEGF blockade and resultant re-opening of SECs fenestrations. This study not only uncovered an indispensible role for VEGF in maintaining structure and function of mature SECs, but also highlights the vasculo-centric nature of PH pathogenesis. Unprecedented ability to rescue PH and its secondary manifestations via manipulating a single vascular factor may also be harnessed for examining the potential utility of de-capillarization treatment modalities.
Authors: Sandhya Gopalakrishnan, Edward N. Harris.
Published: 11-10-2011
The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2. SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7. With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11. In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.
21 Related JoVE Articles!
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Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts
Authors: Jan M. Knaak, Vinzent N. Spetzler, Nicolas Goldaracena, Kristine S. Louis, Nazia Selzner, Markus Selzner.
Institutions: Toronto General Hospital, Toronto General Hospital, Toronto General Hospital.
The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation. These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past. In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved. This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.
Medicine, Issue 90, ex vivo liver perfusion, marginal grafts, DCD
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A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study
Authors: Zhongshu Tang, Fan Zhang, Yang Li, Pachiappan Arjunan, Anil Kumar, Chunsik Lee, Xuri Li.
Institutions: National Eye Institute.
A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.
Medicine, Issue 54, mouse cornea pocket assay, angiogenesis
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Use of a Hanging-weight System for Liver Ischemia in Mice
Authors: Michael Zimmerman, Eunyoung Tak, Maria Kaplan, Mercedes Susan Mandell, Holger K. Eltzschig, Almut Grenz.
Institutions: University of Colorado, Denver, University of Colorado, Denver.
Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.
Medicine, Issue 66, Physiology, Immunology, targeted gene deletion, murine model, liver failure, ischemia, reperfusion, video demonstration
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Rat Mesentery Angiogenesis Assay
Authors: Klas C. Norrby.
Institutions: University of Gothenburg.
The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is translucent and framed by fatty tissue, giving it a window-like appearance. The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; (iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly. Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion. The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated. Review papers (invited) discussing and appraising the assay Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006). Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).
Physiology, Issue 52, angiogenesis, mesentery, objective variables, morphometry, rat, local effects, systemic effects
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Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model
Authors: Kosuke Tsukada, Makoto Suematsu.
Institutions: Keio University, Keio University, Japan Science and Technology Agency (JST).
There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.
Medicine, Issue 66, Physics, Biochemistry, Immunology, Physiology, microcirculation, liver, blood flow, oxygen consumption, phosphorescence, hepatitis
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
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Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice
Authors: Chichi Xie, Weiwei Wei, Tao Zhang, Olaf Dirsch, Uta Dahmen.
Institutions: Jena University Hospital, Jena University Hospital, The First Affiliated Hospital of Wenzhou Medical University.
The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory rate and heart rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure. This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41±2.94 cmH2O which was significantly higher (P<0.05) than before resection (6.87±2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.
Medicine, Issue 92, mice, hemodynamics, hepatic perfusion, CAP, CVP, surgery, intraoperative monitoring, portal vein pressure, blood flow
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Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer
Authors: Elizabeth S. Nakasone, Hanne A. Askautrud, Mikala Egeblad.
Institutions: Watson School of Biological Sciences, Cold Spring Harbor Laboratory, University of Oslo and Oslo University Hospital.
The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.
Cancer Biology, Issue 73, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Genetics, Oncology, Pharmacology, Surgery, Tumor Microenvironment, Intravital imaging, chemotherapy, Breast cancer, time-lapse, mouse models, cancer cell death, stromal cell migration, cancer, imaging, transgenic, animal model
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Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
Authors: Wen-Chi Chen, Sung-Hyun Park, Carol Hoffman, Cecil Philip, Linda Robinson, James West, Gabriele Grunig.
Institutions: New York University School of Medicine, Tuxedo, Vanderbilt University Medical Center, New York University School of Medicine.
The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1. Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system. Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues. The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.
Immunology, Issue 71, Medicine, Anatomy, Physiology, Cardiology, Surgery, Cardiovascular Abnormalities, Inflammation, Respiration Disorders, Immune System Diseases, Cardiac physiology, mouse, pulmonary hypertension, right heart function, lung immune response, lung inflammation, lung remodeling, catheterization, mice, tissue, animal model
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Seven Steps to Stellate Cells
Authors: Patrick Maschmeyer, Melanie Flach, Florian Winau.
Institutions: Harvard Medical School.
Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.
Immunology, Issue 51, Hepatic Stellate Cell, Ito Cell, Liver Immunology, Retinoic Acid, Cell Isolation
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In Vivo Modeling of the Morbid Human Genome using Danio rerio
Authors: Adrienne R. Niederriter, Erica E. Davis, Christelle Golzio, Edwin C. Oh, I-Chun Tsai, Nicholas Katsanis.
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
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Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction
Authors: Kazuyuki Nagai, Shintaro Yagi, Shinji Uemoto, Rene H. Tolba.
Institutions: RWTH-Aachen University, Kyoto University .
Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model. While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14. In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.
Medicine, Issue 73, Biomedical Engineering, Anatomy, Physiology, Immunology, Surgery, liver transplantation, liver, hepatic, partial, orthotopic, split, rat, graft, transplantation, microsurgery, procedure, clinical, technique, artery, arterialization, arterialized, anastomosis, reperfusion, rat, animal model
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Isolation of CD133+ Liver Stem Cells for Clonal Expansion
Authors: C. Bart Rountree, Wei Ding, Hein Dang, Colleen VanKirk, Gay M. Crooks.
Institutions: Pennsylvania State College of Medicine, Pennsylvania State College of Medicine, University of California Los Angeles, School of Medicine.
Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.
Developmental Biology, Issue 56, CD133, liver stem cell, oval cell, liver cancer stem cell, stem cell, cell isolation, non-parenchymal fraction of liver, flow cytometry
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A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics
Authors: Kelly Burrell, Sameer Agnihotri, Michael Leung, Ralph DaCosta, Richard Hill, Gelareh Zadeh.
Institutions: Hospital for Sick Children, Toronto Medical Discovery Tower, Princess Margaret Hospital, Toronto Western Hospital.
We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue. Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules. Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed. Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 μm from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.
Cancer Biology, Issue 76, Medicine, Biomedical Engineering, Cellular Biology, Molecular Biology, Genetics, Neuroscience, Neurobiology, Biophysics, Anatomy, Physiology, Surgery, Intracranial Window, In vivo imaging, Stereotactic radiation, Bone Marrow Derived Cells, confocal microscopy, two-photon microscopy, drug-cell interactions, drug kinetics, brain, imaging, tumors, animal model
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The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye
Authors: Amy E. Birsner, Ofra Benny, Robert J. D'Amato.
Institutions: Boston Children's Hospital, The Hebrew University of Jerusalem, Harvard Medical School.
The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.
Neuroscience, Issue 90, Angiogensis, neovasculatization, in vivo assay, model, fibroblast growth factor, vascular endothelial growth factor
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Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia
Authors: Yves Harder, Daniel Schmauss, Reto Wettstein, José T. Egaña, Fabian Weiss, Andrea Weinzierl, Anna Schuldt, Hans-Günther Machens, Michael D. Menger, Farid Rezaeian.
Institutions: Technische Universität München, University Hospital of Basel, University of Saarland, University Hospital Zurich.
Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.
Medicine, Issue 93, flap, ischemia, microcirculation, angiogenesis, skin, necrosis, inflammation, apoptosis, preconditioning, persistent ischemia, in vivo model, muscle.
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A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress
Authors: Shishir Shetty, Christopher J. Weston, David H. Adams, Patricia F. Lalor.
Institutions: University of Birmingham.
Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this 'adhesion cascade' can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.
Immunology, Issue 85, Leucocyte trafficking, liver, hepatic sinusoidal endothelial cells, peripheral blood lymphocytes, flow adhesion assay
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Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Authors: Ayman I. Omar.
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1. Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10. There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
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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),
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Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler
Authors: Ingmar Königsrainer, Silvio Nadalin, Alfred Königsrainer.
Institutions: Tübingen University Hospital.
Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6 However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.
Medicine, Issue 35, Liver resection, liver tumour, intrahepatic hilus stapling, right hemipedicle
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Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III
Authors: Luigi Boni, Gianlorenzo Dionigi, Francesca Rovera, Matteo Di Giuseppe.
Institutions: University of Insubria, University of Insubria.
Caroli's disease is defined as a abnormal dilatation of the intra-hepatica bile ducts: Its incidence is extremely low (1 in 1,000,000 population) and in most of the cases the whole liver is interested and liver transplantation is the treatment of choice. In case of dilatation limited to the left or right lobe, liver resection can be performed. For many year the standard approach for liver resection has been a formal laparotomy by means of a large incision of abdomen that is characterized by significant post-operatie morbidity. More recently, minimally invasive, laparoscopic approach has been proposed as possible surgical technique for liver resection both for benign and malignant diseases. The main benefits of the minimally invasive approach is represented by a significant reduction of the surgical trauma that allows a faster recovery a less post-operative complications. This video shows a case of Caroli s disease occured in a 58 years old male admitted at the gastroenterology department for sudden onset of abdominal pain associated with fever (>38C° ), nausea and shivering. Abdominal ultrasound demonstrated a significant dilatation of intra-hepatic left sited bile ducts with no evidences of gallbladder or common bile duct stones. Such findings were confirmed abdominal high resolution computer tomography. Laparoscopic left sectoriectomy was planned. Five trocars and 30° optic was used, exploration of the abdominal cavity showed no adhesions or evidences of other diseases. In order to control blood inflow to the liver, vascular clamp was placed on the hepatic pedicle (Pringle s manouvre), Parenchymal division is carried out with a combined use of 5 mm bipolar forceps and 5 mm ultrasonic dissector. A severely dilated left hepatic duct was isolated and divided using a 45mm endoscopic vascular stapler. Liver dissection was continued up to isolation of the main left portal branch that was then divided with a further cartridge of 45 mm vascular stapler. At his point the left liver remains attached only by the left hepatic vein: division of the triangular ligament was performed using monopolar hook and the hepatic vein isolated and the divided using vascular stapler. Haemostatis was refined by application of argon beam coagulation and no bleeding was revealed even after removal of the vascular clamp (total Pringle s time 27 minutes). Postoperative course was uneventful, minimal elevation of the liver function tests was recorded in post-operative day 1 but returned to normal at discharged on post-operative day 3.
Medicine, Issue 24, Laparoscopy, Liver resection, Caroli's disease, Left sectoriectomy
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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