Name: analysis of oxidative stress in zebrafish embryos
Uploaded: 2014-07-07T13:00:00
Description: Watch this Scientific Journal Video about Analysis of Oxidative Stress in Zebrafish Embryos at JoVE.com

Support in Massimo Santoro lab come from HFSP, Marie Curie Action, Telethon and AIRC. We thank Dafne Gays and Emiliano Panieri for critical reading of the manuscript.

1. Preparation of Instruments and Working Solutions
<ol>
	<li>Prepare the fish water solution. Make a stock solution by dissolving 2 g of sea salts &#39;Instant Ocean&#39; in 50 ml of distilled water. Add 1.5 ml of stock fish water to 1 L distilled water to prepare ready to use fish water (60 &#181;g/ml sea salts final concentration). Autoclave the ready to use fish water before usage. This solution is used as zebrafish embryo medium.</li>
	<li>Prepare methylcellulose for embryo mounting. Dissolve 1.5 g of methylcellul...

Critical Steps
The procedure for oxidative stress detection in zebrafish embryos herein described comprises two different methods. The whole mount ROS-detection method is mainly a qualitative assay for ROS-detection, while the single cell ROS-detection method allows more specific quantitative measurements (Figure 1). Both methods offer a quick and easy way to assess in vivo ROS-detection on zebrafish embryos. However, they both present some critical steps.
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Oxidative stress is specifically defined as a condition that results from an unbalanced cellular redox state. The complex redox reactions that routinely occur inside cells determine the cellular redox-state. Redox reactions consist of all chemical reactions that consist in the transfer of electrons between atoms of biological molecules producing reduction and oxidation of molecules (i.e. redox reactions). These reactions are catalyzed by electronically activated species (i.e. pro-oxidative species), which are characterized by an extreme structural instability and spontaneous activation of unbalanced electrons that exchange with neighboring biomolecules. These irregular reactions result into DNA damage, protein carboxylation, and lipid oxidation, and eventually lead to cell death1. Increased levels of oxidative stress have been associated with aging and the progression of different pathological states2. Oxidative stress has been reported to be responsible for vascular alterations in diabetes and cardiovascular diseases3,4. It also plays a critical role in neuronal degeneration in Alzheimer&#39;s disease and Parkinson&#39;s disease5. Moreover, oxidative stress has been demonstrated as a critical factor in governing cancer progression and metastatic events6,7. In addition, inflammation and immune responses may elicit and further support oxidative stress8.
In living cells, pro-oxidative species are derived from oxygen (ROS; reactive oxygen species) or nitrogen (RNS; reactive nitrogen species). ROS include the hydroxyl radical (.OH), the superoxide anion (O2-), and the hydrogen peroxide (H2O2). The primary RNS is nitrous oxide (NO.). A series of secondary reactive species can be generated by spontaneous interactions between ROS and RNS or free metals ions9. For example, the superoxide anion reacts with nitrous oxide to form peroxynitrate (ONOO-), while H2O2 reacting with Fe2+ generates hydroxyl radicals. ROS and RNS, due to their ability to react with several biomolecules, are considered a dangerous threat for the maintenance of the physiological redox state10. To maintain the redox state cells are equipped with a series of detoxifying anti-oxidant molecules and enzymes. The superoxide dismutase (SOD), Catalase, Glutathione peroxidase and Peroxiredoxins essentially constitute the anti-oxidant enzymatic-arsenal that provides cellular protection from pro-oxidative species including H2O2 , .OH and OONO- 11. Also anti-oxidant molecules like vitamin C and E, polyphenols and CoenzymeQ10 (CoQ10) are of critical importance to quench ROS and their dangerous derivatives12,13. However, an excessive production of ROS and RNS, or a dysfunction in the anti-oxidant system, shifts the cellular redox-state toward oxidative stress14.
Besides their negative connotation, ROS can play various physiological roles in cells of different origin. Cells normally produce ROS as signaling molecules to mediate normal biological events such as host defense and wound repair15-17. Reactive species are normally produced in cells by intracellular enzymes such as NOX (NADPH Oxidase) and XO (Xantine Oxidase) in response to signaling factors, growth factors, and intracellular fluctuations of calcium levels18,19. It has been reported that ROS may differentially modulate the activity of important nuclear factors such as p53 or cellular components such as the ATM-kinase, a master regulator of the response to DNA damage20. Analogously ROS strongly influence cellular signaling by mediating the oxidation and inactivation of protein tyrosine phosphatases (PTPs), which are established as critical regulators of signal transduction21. Moreover, proteomic based methodologies demonstrate that RNS are also responsible for specific protein modifications and alterations of molecular signaling. RNS react with the cysteine thiol groups modifying them into S-nitrothiols (SNO) and triggering molecular pathways concomitant with pathological states such as inflammatory and autoimmune diseases22,23.
Since cell culture experiments only partially reproduce the multitude of factors acting in vivo, it is of great interest to perform redox studies in animal models24,25. To achieve this, the zebrafish has been considered a suitable vertebrate animal model to study oxidative stress dynamics26. The zebrafish is a new model system that grants several advantages to study cellular and genetic events during vertebrate development and disease. Large clusters of embryos can be generated and available weekly for experimental needs. Moreover the extraordinary optical clarity of zebrafish embryos, as well their small size, enables single cell imaging and dynamic tracking in a whole organisms27. In the last decade, a considerable number of zebrafish mutants have been generated to model human pathological conditions such as cancer and genetic diseases28-31. Most importantly, a multitude of transgenic lines has been produced to allow extensive opportunities of genetic and biological manipulations32. For example, transgenic tissue-specific zebrafish lines are regularly utilized for in vivo studies. These lines express a fluorescent protein under the control of a selected promoter, offering the ability to identify single cells in vivo, as well as the anatomical structure they comprise.
Several toxicological studies have already used the zebrafish to evaluate the in vivo effect of chemicals on redox homeostasis, suggesting the suitability of this vertebrate as an animal model for the field of drug discovery and oxidative stress33-35. Even though some fluorescent probes have been tested to monitor oxidative stress in zebrafish larvae36,37, there are no established assays to detect and measure the levels of oxidative stress in zebrafish tissues and living cells. Here we describe a procedure for in vivo quantification of oxidative stress in living cells of zebrafish embryos. Imaging tools, FACS sorting, fluorescent probes and pro-oxidative conditions are all combined to generate a simple assay for the detection and quantification of oxidative species in zebrafish embryos and tissues.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:285px;">Catalog Number</td>
			<td style="width:528px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hydrogen peroxide solution</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">516813</td>
			<td>DO NOT STORE DILUITIONS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hank&#39;s Balanced Salt Solution 1X</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">14025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Methyl cellulose</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">M0387</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Instant Ocean Aquarium Sea Salt Mixture</td>
			<td style="width:147px;">INSTANT OCEAN</td>
			<td style="width:285px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricaine</td>
			<td style="width:147px;">SIGMA</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Cgeneric ROS-sensitive probe:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; CellROX Deep Red Reagent</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">C10422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Mitochondria specific ROS-sensitive probe: MitoSOX&#160;</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">M36008</td>
			<td>dissolve one vial with 13&#956;l of DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hydroethidine</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">D23107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Rotenone</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">R8875</td>
			<td>Prepare 5mM stock solution in DMSO.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Dimethyl sulfoxide</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">D2650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">VAS2870; 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine</td>
			<td style="width:147px;">EnzoLifeScience</td>
			<td>BML-EI395</td>
			<td>dissolve the powder in DMSO; diluite in fish water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Propidium Iodide&#160;</td>
			<td style="width:147px;">Molecular probes&#160;&#160;&#160;&#160;&#160;&#160; (Life Technologies)&#160;</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">7-aminoactinomycin D (7-AAD)&#160;</td>
			<td style="width:147px;">Molecular probes&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (Life Technologies)&#160;</td>
			<td>A1310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Nrf2a Morpholino</td>
			<td style="width:147px;">GeneTools</td>
			<td style="width:285px;">5&#39;-CATTTCAATCTCCATCATGTCTCAG-3&#39;</td>
			<td>Ref: Timme-LaLaragy et al; 2012 (PMID: 22174413); Kobayashi et al; 2002(PMID:12167159 )</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td style="width:147px;">ROCHE</td>
			<td>11213857001</td>
			<td>Dissolve the powder at 100mg/ml in sterile HBSS. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">10010-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum&#160;</td>
			<td style="width:147px;">GIBCO</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:147px;">ROCHE</td>
			<td style="width:285px;"></td>
			<td>Dissolve one tablet in 1ml of water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin-EDTA (10x), no phenol red</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">15400-054</td>
			<td>Prepare 1X working solution before usage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Compound microscope&#160;</td>
			<td>ZEISS</td>
			<td style="width:285px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereo microscope with fluorescent illumination</td>
			<td>Nikon</td>
			<td style="width:285px;">AZ100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">camera&#160;</td>
			<td>ZEISS</td>
			<td style="width:285px;">AxioCamMRm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">software for fluorescence image acquisition</td>
			<td>ZEISS</td>
			<td style="width:285px;">ZEN 2011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence-activated cell sorter</td>
			<td style="width:147px;">BD FACSCalibur</td>
			<td style="width:285px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge&#160;</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:285px;">5417R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes&#160;</td>
			<td style="width:147px;">BD</td>
			<td>342065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell Plate&#160;</td>
			<td>BD Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Sterilized, non treated Petri dishes 90mm</td>
			<td style="width:147px;">VWR</td>
			<td style="width:285px;">391-1915</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Confocal microscope</td>
			<td style="width:147px;">Leica</td>
			<td style="width:285px;">Leica SP5</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of oxidative stress in zebrafish embryos

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 &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; 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.

By applying the method here described, we can easily measure and detect oxidative stress (and ROS levels) in zebrafish embryonic tissues. After crossing adult zebrafish, eggs are collected and allowed to develop at 28 &#176;C to 72 hr&#160;post fertilization (hpf). In order to induce oxidative stress, we propose two different approaches: 1) the treatment of embryos with strong pro-oxidant reagents or 2) promoting ROS formation after tissue injury.
In the first approach, we employed two differe...

Watch this Scientific Journal Video about Analysis of Oxidative Stress in Zebrafish Embryos at JoVE.com

Analysis of Oxidative Stress in Zebrafish Embryos

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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