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TXNL6 is a novel oxidative stress-induced reducing system for methionine sulfoxide reductase a repair of ?-crystallin and cytochrome C in the eye lens.
PUBLISHED: 07-27-2010
A key feature of many age-related diseases is the oxidative stress-induced accumulation of protein methionine sulfoxide (PMSO) which causes lost protein function and cell death. Proteins whose functions are lost upon PMSO formation can be repaired by the enzyme methionine sulfoxide reductase A (MsrA) which is a key regulator of longevity. One disease intimately associated with PMSO formation and loss of MsrA activity is age-related human cataract. PMSO levels increase in the eye lens upon aging and in age-related human cataract as much as 70% of total lens protein is converted to PMSO. MsrA is required for lens cell maintenance, defense against oxidative stress damage, mitochondrial function and prevention of lens cataract formation. Essential for MsrA action in the lens and other tissues is the availability of a reducing system sufficient to catalytically regenerate active MsrA. To date, the lens reducing system(s) required for MsrA activity has not been defined. Here, we provide evidence that a novel thioredoxin-like protein called thioredoxin-like 6 (TXNL6) can serve as a reducing system for MsrA repair of the essential lens chaperone ?-crystallin/sHSP and mitochondrial cytochrome c. We also show that TXNL6 is induced at high levels in human lens epithelial cells exposed to H(2)O(2)-induced oxidative stress. Collectively, these data suggest a critical role for TXNL6 in MsrA repair of essential lens proteins under oxidative stress conditions and that TXNL6 is important for MsrA defense protection against cataract. They also suggest that MsrA uses multiple reducing systems for its repair activity that may augment its function under different cellular conditions.
Authors: Konstantin Galichanin, Nooshin Talebizadeh, Per Söderberg.
Published: 11-28-2012
Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental 2-4 and epidemiological evidence 5,6 that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized 7 and non-anesthetized animals 8. The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% 9. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals 10. Our focus has recently been apoptosis in the lens as the mechanism for cataract development 8,11,12. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract. In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.
24 Related JoVE Articles!
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A Novel Technique of Rescuing Capsulorhexis Radial Tear-out using a Cystotome
Authors: Shah M. R. Karim, Chin T. Ong, Mizanur R. Miah, Tamsin Sleep, Abdul Hanifudin.
Institutions: Hairmyres Hospital, NHS Lanarkshire, Royal Devon and Exeter NHS Foundation Trust, National Institute of Ophthalmology, South Devon Healthcare NHS Trust.
Part 1 : Purpose: To demonstrate a capsulorhexis radial tear out rescue technique using a cystotome on a virtual reality cataract surgery simulator and in a human eye. Part 2 : Method: Steps: When a capsulorhexis begins to veer radially towards the periphery beyond the pupillary margin the following steps should be applied without delay. 2.1) Stop further capsulorhexis manoeuvre and reassess the situation. 2.2) Fill the anterior chamber with ophthalmic viscosurgical device (OVD). We recommend mounting the cystotome to a syringe containing OVD so that the anterior chamber can be reinflated rapidly. 2.3) The capsulorhexis flap is then left unfolded on the lens surface. 2.4) The cystotome tip is tilted horizontally to avoid cutting or puncturing the flap and is engaged on the flap near the leading edge of the tear but not too close to the point of tear. 2.5) Gently push or pull the leading edge of tear opposite to the direction of tear. 2.6) The leading tearing edge will start to do a 'U-Turn'. Maintain the tension on the flap until the tearing edge returns to the desired trajectory. Part 3 : Results: Using our technique, a surgeon can respond instantly to radial tear out without having to change surgical instruments. Changing surgical instruments at this critical stage runs a risk of further radial tear due to sudden shallowing of anterior chamber as a result of forward pressure from the vitreous. Our technique also has the advantage of reducing corneal wound distortion and subsequent anterior chamber collapse. Part 4 : Discussion The EYESI Surgical Simulator is a realistic training platform for surgeons to practice complex capsulorhexis tear-out techniques. Capsulorhexis is the most important and complex part of phacoemulsification and endocapsular intraocular lens implantation procedure. A successful cataract surgery depends on achieving a good capsulorhexis. During capsulorhexis, surgeons may face a challenging situation like a capsulorhexis radial tear-out. A surgeon must learn to tackle the problem promptly without making the situation worse. Some other methods of rescuing the situation have been described using a capsulorhexis forceps. However, we believe our method is quicker, more effective and easier to manipulate as demonstrated on the EYESi surgical simulator and on a human eye. Acknowledgments: List acknowledgements and funding sources. We would like to thank Dr. Wael El Gendy, for video clip. Disclosures: describe potential conflicting interests or state We have nothing to disclose. References: 1. Brian C. Little, Jennifer H. Smith, Mark Packer. J Cataract Refract Surg 2006; 32:1420 1422, Issue-9. 2. Neuhann T. Theorie und Operationstechnik der Kapsulorhexis. Klin Monatsbl Augenheilkd. 1987; 1990: 542-545. 3. Gimbel HV, Neuhann T. Development, advantages and methods of the continuous circular capsulorhexis technique. J Cataract Refract Surg. 1990; 16: 31-37. 4. Gimbel HV, Neuhann T. Continuous curvilinear capsulorhexis. (letter) J Cataract Refract Sur. 1991; 17: 110-111.
Medicine, Issue 47, Phacoemulsification surgery, cataract surgery, capsulorhexis, capsulotomy, technique, Continuous curvilinear capsulorhexis, cystotome, capsulorhexis radial tear, capulorhexis COMPLICATION
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Intravitreous Injection for Establishing Ocular Diseases Model
Authors: Kin Chiu, Raymond Chuen-Chung Chang, Kwok-Fai So.
Institutions: The University of Hong Kong - HKU.
Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given. To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45° injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.
Neuroscience, Issue 8, eye, injection, rat
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A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt
Authors: Rital B. Bhavsar, Kenta Nakamura, Panagiotis A. Tsonis.
Institutions: University of Dayton, University of Dayton.
Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.
Cellular Biology, Issue 52, IPE cells, lens, regeneration, newt
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Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
Authors: Jessica M. Skeie, Stephen H. Tsang, Vinit B. Mahajan.
Institutions: University of Iowa, University of Iowa, Columbia University College of Physicians and Surgeons.
While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.
Cellular Biology, Issue 50, mouse, vitreous, retina, proteomics, superoxide dismutase
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Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers
Authors: Curtis C. Hughey, Dustin S. Hittel, Virginia L. Johnsen, Jane Shearer.
Institutions: University of Calgary, University of Calgary.
Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis. The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture. This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.
Physiology, Issue 48, cardiac fibers, mitochondria, oxygen consumption, mouse, methodology
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A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
Authors: Kerstin Trompelt, Janina Steinbeck, Mia Terashima, Michael Hippler.
Institutions: University of Münster, Carnegie Institution for Science.
The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.
Microbiology, Issue 85, Sucrose density gradients, Chlamydomonas, multiprotein complexes, 15N metabolic labeling, thylakoids
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CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
Authors: Jing Ge, Somsak Prasongtanakij, David K. Wood, David M. Weingeist, Jessica Fessler, Panida Navasummrit, Mathuros Ruchirawat, Bevin P. Engelward.
Institutions: Massachusetts Institute of Technology, Chulabhorn Graduate Institute, University of Minnesota.
DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The ‘CometChip’ is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.
Bioengineering, Issue 92, comet assay, electrophoresis, microarray, DNA damage, DNA repair
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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Authors: Grażyna Nowak, Diana Bakajsova.
Institutions: University of Arkansas for Medical Sciences .
The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC. Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis. These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.
Cellular Biology, Issue 71, Biochemistry, Molecular Biology, Genetics, Pharmacology, Physiology, Medicine, Protein, Mitochondrial dysfunction, mitochondria, protein kinase C, renal proximal tubular cells, reactive oxygen species, oxygen consumption, electron transport chain, respiratory complexes, ATP, adenovirus, primary culture, ischemia, cells, flow cytometry
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Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
Authors: Rami R. Ajjuri, Janis M. O'Donnell.
Institutions: University of Alabama.
Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.
Immunology, Issue 82, biology (general), environmental effects (biological, animal and plant), immunology, animal models, Immune System Diseases, Pathological Conditions, Signs and Symptoms, Life Sciences (General), Neuroinflammation, inflammation, nitric oxide, nitric oxide synthase, Drosophila, neurodegeneration, brain, Griess assay, nitrite detection, innate immunity, Parkinson disease, tissue culture
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A Novel Capsulorhexis Technique Using Shearing Forces with Cystotome
Authors: Shah M. R. Karim, Chin T. Ong, Tamsin J. Sleep.
Institutions: Hairmyres Hospital, NHS Lanarkshire, Department of Ophthalmology, South Devon Healthcare NHS Trust.
Purpose: To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye. Method: Our technique involves creating the initial anterior capsular tear with a cystotome to raise a flap. The flap left unfolded on the lens surface. The cystotome tip is tilted horizontally and is engaged on the flap near the leading edge of the tear. The cystotome is moved in a circular fashion to direct the vector forces. The loose flap is constantly swept towards the centre so that it does not obscure the view on the tearing edge. Results: Our technique has the advantage of reducing corneal wound distortion and subsequent anterior chamber collapse. The capsulorhexis flap is moved away from the tear leading edge allowing better visualisation of the direction of tear. This technique offers superior control of the capsulorhexis by allowing the surgeon to change the direction of the tear to achieve the desired capsulorhexis size. Conclusions: The EYESI Surgical Simulator is a realistic training platform for surgeons to practice complex capsulorhexis techniques. The shearing forces technique is a suitable alternative and in some cases a far better technique in achieving the desired capsulorhexis.
JoVE Medicine, Issue 39, Phacoemulsification surgery, cataract surgery, capsulorhexis, capsulotomy, technique, Continuous curvilinear capsulorhexis, cystotome
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Authors: Nikki M. Curthoys, Michael J. Mlodzianoski, Dahan Kim, Samuel T. Hess.
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
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Analysis of Oxidative Stress in Zebrafish Embryos
Authors: Vera Mugoni, Annalisa Camporeale, Massimo M. Santoro.
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
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Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells
Authors: Jason D. Vevea, Dana M. Alessi Wolken, Theresa C. Swayne, Adam B. White, Liza A. Pon.
Institutions: Columbia University, Columbia University.
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
Bioengineering, Issue 77, Microbiology, Cellular Biology, Molecular Biology, Biochemistry, life sciences, roGFP, redox-sensitive green fluorescent protein, GO-ATeam, ATP, FRET, ROS, mitochondria, biosensors, GFP, ImageJ, microscopy, confocal microscopy, cell, imaging
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Viability Assays for Cells in Culture
Authors: Jessica M. Posimo, Ajay S. Unnithan, Amanda M. Gleixner, Hailey J. Choi, Yiran Jiang, Sree H. Pulugulla, Rehana K. Leak.
Institutions: Duquesne University.
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.
Cellular Biology, Issue 83, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
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Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer
Authors: Huy Nguyen, Cristy Loustaunau, Alexander Facista, Lois Ramsey, Nadia Hassounah, Hilary Taylor, Robert Krouse, Claire M. Payne, V. Liana Tsikitis, Steve Goldschmid, Bhaskar Banerjee, Rafael F. Perini, Carol Bernstein.
Institutions: University of Arizona, Tucson, Tucson, AZ, University of Arizona, Tucson, Tucson, AZ, University of Arizona, Tucson.
In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia. Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer. DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia. We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.
Cellular Biology, Issue 41, DNA Repair, Apoptosis, Field Defect, Colon Cancer, Pms2, ERCC1, Cytochrome c Oxidase Subunit I, Ku86, Immunohistochemistry, Cancer Resection
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Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Authors: Ido Karady, Anna Frumkin, Shiran Dror, Netta Shemesh, Nadav Shai, Anat Ben-Zvi.
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
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Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development
Authors: Chryssostomos Chatgilialoglu, Carla Ferreri, Annalisa Masi, Michele Melchiorre, Anna Sansone, Michael A. Terzidis, Armida Torreggiani.
Institutions: Consiglio Nazionale delle Ricerche.
The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.
Chemistry, Issue 74, Biochemistry, Chemical Engineering, Chemical Biology, chemical analysis techniques, chemistry (general), life sciences, radiation effects (biological, animal and plant), biomarker, biomimetic chemistry, free radicals, trans lipids, cyclopurine lesions, DNA, chromatography, spectroscopy, synthesis
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Methods to Assess Subcellular Compartments of Muscle in C. elegans
Authors: Christopher J. Gaffney, Joseph J. Bass, Thomas F. Barratt, Nathaniel J. Szewczyk.
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
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Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Authors: Melissa N. Patterson, Patrick H. Maxwell.
Institutions: Rensselaer Polytechnic Institute.
Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
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Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Authors: Kristen K. McCampbell, Kristin N. Springer, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
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Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Authors: Jennifer C. Arnold, Michael F. Salvatore.
Institutions: Louisiana State University Health Sciences Center.
There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
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Proper Care and Cleaning of the Microscope
Authors: Victoria Centonze Frohlich.
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.
Basic Protocols, Issue 18, Current Protocols Wiley, Microscopy, Cleaning the Microscope
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Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
Authors: Peggy S. Zelenka, Chun Y. Gao, Senthil S. Saravanamuthu.
Institutions: National Eye Institute (NEI), National Institutes of Health (NIH).
The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body. Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens. Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2. Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers. The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied. Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques. Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.
Cellular Biology, Issue 31, lens, differentiation, FGF, rat
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Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants
Authors: Yan Zhang, Kyle McCulloch, Jarema Malicki.
Institutions: The Second Teaching Hospital of Jilin University, Harvard Medical School.
The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.
Developmental Biology, Issue 28, Zebrafish, lens mutation, lens transplantation
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