Analysis of Oxidative Stress in Zebrafish Embryos

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 diseases^3,4. It also plays a critical role in neuronal degeneration in Alzheimer's disease and Parkinson's disease⁵. Moreover, oxidative stress has been demonstrated as a critical factor in governing cancer progression and metastatic events^6,7. 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 derivatives^12,13. 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 repair^15-17. 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 levels^18,19. 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 diseases^22,23.

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 models^24,25. 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 diseases^28-31. 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 stress^33-35. Even though some fluorescent probes have been tested to monitor oxidative stress in zebrafish larvae^36,37, 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.

Protokół

1. Preparation of Instruments and Working Solutions

1. Prepare the fish water solution. Make a stock solution by dissolving 2 g of sea salts 'Instant Ocean' 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 µg/ml sea salts final concentration). Autoclave the ready to use fish water before usage. This solution is used as zebrafish embryo medium.
2. Prepare methylcellulose for embryo mounting. Dissolve 1.5 g of methylcellulose in 50 ml of sterile fish water. Facilitate the dissolution by using a magnet on a stir plate. The complete dissolution of the powder may require several hours. Check the solution for clarity and aliquot into small tubes. Store at -20 °C for months and thaw out aliquots at use. Centrifuge the methylcellulose at 950 x g for 5 min before using. Avoid freeze-thaw cycles of aliquots.
3. Prepare 50 ml of tricaine/ethyl 3-aminobenzoate methanesulphonate salt (stock solution) by dissolving 200 mg of tricaine in 100 ml water and adjust pH to 7.0 using Tris-HCl 1 M (pH 9). Store this stock at 4 °C for no more than 30 days. CAUTION: tricaine is toxic. Use in accordance with appropriate handling guidelines.
4. Inducing oxidative stress in zebrafish embryos
   1. Prepare an oxidant solution for generic oxidative stress induction: Make 50 ml of oxidant solution by adding H₂O₂ stock solution (hydrogen peroxide; 100 mM) to fish water. Use H₂O₂ of a final concentration between 2 mM and 100 μM. Prepare this solution shortly before usage. The oxidant solution can be applied to both whole mount ROS-detection and single-cell ROS-detection methods. Do not store this solution. CAUTION: H₂O₂ is dangerous, and harmful by inhalation and if swallowed. Contact with combustible material may cause fire. Handle under a fume hood and wear appropriate personal protective equipment.
   2. Prepare an oxidant solution for mitochondria-derived oxidative stress induction: Make a oxidant stock solution (5 mM) by dissolving 3.9 mg of rotenone in 2 ml of dimethyl sulfoxide (DMSO). Keep this solution at room temperature in the dark.
      1. Dissolve rotenone stock solution to 10 ml of fish water to make a ready to use solution. Use rotenone at a concentration between 5-50 μM. Do not use rotenone at concentrations higher than 100 μM. At appropriate concentrations, this oxidant solution can be applied to both whole mount ROS-detection and single-cell ROS-detection methods. CAUTION: Rotenone is toxic and hazardous. Handle according appropriate precautionary statements.
   3. Induce oxidative stress by gene knock-down: Knock-down nrf2a gene expression in zebrafish embryos by morpholino microinjection as previously reported by Timme-Laragy A. et al., 2012³⁸.
   4. Induce oxidative stress after tissue damage: generate a wound margin at the tail fin of zebrafish embryos at 72 hpf as previously described by Niethammer et al., 2009³⁹.
5. Prepare 5 ml of ROS-detection solution for single cell ROS-detection method:
   1. Solution for general ROS detection: Dissolve the generic ROS-sensitive molecular probe in HBSS (Hanks’s Balanced Salt Solution) in order to prepare a working solution (concentration range: 2.5-10 μM). This solution must be prepared shortly before usage.
   2. Solution for specific detection ROS induced by mitochondria: Shortly before use, dissolve a mitochondrial ROS-sensitive probe with DMSO making a 5 mM stock solution. Dissolve stock solution in HBSS in order to prepare a working solution (concentration range: 2.5-10 μM).
      NOTE: Avoid light and oxygen exposure of ROS-detection working solutions and their respective stock solutions. Protect the tube from light by using an aluminum foil. Do not store or re-use probes dissolved in HBSS. Store the stock solutions at -20 °C for a month.
      CAUTION: Handle molecular probes and DMSO under a fume hood in accordance with appropriate guidelines.
6. Prepare 5 ml of ROS-detection solution for the whole mount ROS-detection method: Dissolve the generic ROS-sensitive probe stock solution (stabilized in DMSO) in HBSS in order to prepare a working solution (concentration range: 2.5-5 μM). Avoid light and oxygen exposure. Protect the tube from light. Prepare this solution before use. Do not store or re-use this solution. CAUTION: Handle molecular probes under a fume hood in accordance with appropriate guidelines.
7. Make 25 ml of Stop Solution by adding 10 ml of fetal bovine serum to 15 ml of PBS 1x. Keep solution sterile. Store this solution at 4 °C. This solution is required only for single cell - ROS detection method.
8. Set the zebrafish air incubator at 28 °C.
9. Set the centrifuge at 4 °C for the single cell ROS-detection method.

2. Mating of Adult Fishes and Selection of Zebrafish Embryos

1. Set-up adult zebrafish pair crosses according to standard protocols⁴⁰. Select the appropriate transgenic line in accordance with specific experimental needs.
2. Collect eggs and place them into a 90-mm dish with fish water/embryo medium. Keep eggs at 28 °C until embryos will develop and grow to the desired developmental stage (e.g., 48 hpf, 72 hpf; hpf: hours post fertilization).
3. Screen for developing embryos. Exclude all unfertilized eggs or underdeveloped embryos.
4. Anesthetize embryos by adding 1 ml of tricaine (stock solution) in 50 ml fish water.
5. Select Tg fluorescent embryos under a stereomicroscope.

3. Treatment of Embryos with Oxidant Agent

1. Use at least 30 embryos between 48 hpf and 72 hpf per different condition. Wash embryos twice with fresh fish water in order to remove tricaine.
2. Split fluorescent embryos into three dishes. Put no more than 30 embryos/dish.
3. Remove the washing solution and add 10 ml of the oxidant solution or 10 ml of fish water as control solution.
4. Incubate embryos for 10 min to 1 hr at 28 °C. The incubation period depends by the level of oxidative stress to be induced in specific tissues. Short periods are the best as cells affected by oxidative stress progressively undergo cell death.
5. Transfer embryos into a new dish containing HBSS and wash embryos by swirling. Pre-warm HBSS at 28 °C.

4. Whole Mount ROS Detection Method

1. Collect embryos after oxidant treatment and put no more than 10 embryos in a small tube. Rinse with HBSS as much as possible. Protect the tube from light using aluminum foil.
2. Add 1 ml of ROS-detection solution for each tube.
3. Incubate embryos in the dark for 15 min at 28 °C to avoid light exposure.
4. During the incubation time prepare a glass slide for whole embryo analysis. Put 300 μl of methylcellulose on a “depression” glass slide and spread it on the glass surface with a pipette tip. Avoid bubbles while releasing the methylcellulose.
5. At the end of the incubation time, immediately remove the ROS-detection solution and wash twice with 2 ml of HBSS. Wash embryos by inverting the tube several times. Do not pipette or vortex.
6. Aspirate embryos up into a glass Pasteur pipette. Position embryos near the opening of the pipette and gently eject them into the methylcellulose. Orientate the embryos appropriately with a fine nylon line.
7. Compare the fluorescence of control embryos with treated embryos. Alternatively, fix parameters of the fluorescence stereomicroscope or confocal microscope so that all embryos are imaged using the same imaging settings.

5. Single Cell ROS-detection Method

1. Start from step: 3.5. Make sure to have at least 35 embryos for each condition.
2. Transfer embryos into a new dish. Remove fish water as much as possible. Add 10 ml of ice-cold PBS 1x and 400 μl of Protease Inhibitors Cocktail. Manually dechorionate embryos with forceps and remove the yolk sack with a fine needle. Note: Removing yolk sack ensures clean samples for the following FACS analysis. This step can be avoided according to FACS instrument.
3. Transfer de-yolked embryos into a 24-well multiwell plate (15 embryos/well) and remove all fish water.
4. Add 300 μl of HBSS, 30 μl collagenase P, 50 μl Trypsin-EDTA in each well.
5. Homogenize embryos by gently pipetting up and down with a tight pipette tip (1,000 μl).
6. Incubate at 28 °C for 20 min and homogenize samples by pipetting every 5 min.
7. Check tissue disruption by observing an aliquot of homogenate at the compound microscope. Facilitate single cell suspension by pipetting. Do not incubate embryos for more than 30 min.
8. Stop the reaction by adding 200 μl of Stop Solution. Mix by pipetting gently.
9. Transfer cell suspension into a pre-chilled tube with tight bottom. Keep tube on ice and avoid light exposure.
10. Centrifuge for 5 min at 250 x g at 4 °C.
11. Remove the upper phase and re-suspend cells with ice cold HBSS by gently pipetting.
12. Count cells and make sure you have the same number of cells in all of your samples. Do not use less than 2 x 10⁶ cells.
13. Centrifuge cells for 5 min at 250 x g at 4 °C.
14. Remove supernatant and suspend the pellet with 1 ml of ice chilled HBSS containing the ROS-sensitive probe.
15. Incubate the tube at room temperature for 3 min in the dark. Vary incubation time according to the level of oxidative stress and to the sensitivity of the probe.
16. Transfer cells to a FACS tube. Keep tubes on ice and avoid light exposure before FACS analysis.
17. Sort cells with a fluorescence-activated cell sorter (FACS). Set wavelengths in accordance with Tg tissue fluorescence (e.g., GFP) and the excitation/emission spectra of the molecular probe used for ROS detection (e.g., specific mitochondrial ROS-sensitive probes, generic ROS-sensitive probes).
18. For each condition, measure all samples within the same experimental session by applying the same FACS settings. Calculate the percentage of fluorescent cells as the mean of different biological replicates. Analyze at least two replicates for each condition.

Wyniki

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 °C to 72 hr 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...

Dyskusje

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.

Materiały

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