VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

The procedures described in this protocol were approved by the Institutional Animal Care and Use Committee at the Indiana University of Pennsylvania.
1. Harvest Embryos
<ol>
	<li>Maintain zebrafish at 28.5 &#176;C, pH 7, conductivity between 500-1,500 &#181;S, and a light/dark cycle of 14 hr light and 10 hr dark24. Use wild-type strains such as T&#252;, AB or T&#252;/AB hybrid. Different strains may respond differently to chemical treatment25.</li>
	<li>Set up the fish for mating the night before harvesting eggs by adding fish system water into a mating tank. Add a male and female fish into the tank and separate the two fish with a divider. Each fish pair will produce a range of 50-300 eggs. To ensure that enough eggs will be produced, set up 30 pairs of fish. Typically, about 50% of fish pairs at the prime mating age (6-9 months old) will produce eggs, resulting up to 200 eggs per pair and a maximum of up to 3,000 eggs for this experiment.</li>
	<li>The next morning after the light turns on, remove the divider to initiate mating. Check the mating tanks for eggs every 15 min.</li>
	<li>Once the fish lay eggs, harvest all embryos using a tea strainer and combine them into one large container with E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). At 1.5 hpf, remove and discard unfertilized eggs with a plastic transfer pipet under a dissecting microscope.&#160;Unfertilized eggs are opaque while fertilized eggs are transparent34.</li>
	<li>Transfer 50 embryos into a 100 x 15 mm glass Petri dish containing 50 ml E3 buffer for each treatment condition. A total of 33 dishes are needed for 11 treatment conditions and 3 replicates.</li>
</ol>
2. Treating Embryos
<ol>
	<li>Carry out treatments at 2 hr post-fertilization (hpf)35. Incubate embryos/larvae at 28.5 &#176;C and examine the larvae at 120 hpf. Perform at least three replicates of each treatment condition for statistical analysis.</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 300 mM ethanol diluted in E3 buffer. Cover the Petri dishes with Parafilm to minimize evaporation of ethanol.
	<ol>
		<li>Continue exposing the embryos to ethanol for 22 hr. Then remove the ethanol with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the ethanol. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of new E3 buffer.</li>
	<li>For 9 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 1,000 mg/L sodium nitrite dissolved in E3 buffer. Confirm the concentration of the stock nitrite solution beforehand using a modification of the diazotization (USEPA Method 354.1) spectrophotometric method28.
	<ol>
		<li>Continue exposing 3 dishes for 46 hr, 3 dishes for 70 hr and 3 dishes for 94 hr. Replace the nitrite solution with freshly made nitrite solution daily.</li>
		<li>After each exposure time, remove the nitrite with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the nitrite. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 200 mg/L sodium nitrite. Repeat this with 400, 600, 800, and 1,000 mg/L of sodium nitrite.
	<ol>
		<li>Continue exposing the embryos to nitrite for 70 hr. Replace the nitrite solution with freshly made nitrite solution daily.</li>
		<li>After the exposure time, remove the nitrite with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the nitrite. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 1,000 mg/L sodium nitrate dissolved in E3 buffer. Confirm the concentration of the stock nitrate solution beforehand using a modification of the cadmium reduction (USEPA Method 353.3) spectrophotometric method28.
	<ol>
		<li>Continue exposing the embryos to nitrate for 70 hr. Replace the nitrate solution with freshly made nitrate solution daily.</li>
		<li>After the exposure time, remove the nitrate with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the nitrate. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>During each day of the exposure, count the number of dead embryo/larvae using a stereomicroscope. Signs of death include lack of heartbeat and blood circulation, or lack of motility after 1 min of observation. Remove dead embryos/larvae with a transfer pipet to reduce contamination of the E3 buffer.</li>
	<li>When experiments end at 120 hpf, euthanize the larvae.
	<ol>
		<li>Remove the E3 buffer with a transfer pipet. Then add 50 ml of 0.2% MS-222 (buffered to pH 7) and wait for 10 min.</li>
		<li>Remove the MS-222 with a transfer pipet. Add 50 ml of E3 buffer and swirl to wash out the MS-222.</li>
		<li>Remove the E3 buffer with a transfer pipet and add 20 ml of 4% paraformaldehyde (PFA) to fix the larvae. Swirl the dish several times. Use a transfer pipet to transfer the larvae into a glass vial along with enough PFA to fill the vial. Store the vials in the refrigerator overnight (O/N).</li>
	</ol>
	</li>
	<li>Take pictures of fixed larvae using a stereoscope with a digital camera. Use 30X magnification, ISO 200, and 200 msec&#160;exposure time. Orient the larvae so that the anterior to the left and the dorsal is to the top of the field.</li>
</ol>

<ol><li>Gilbert-Barness, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Teratogenic+causes+of+malformations%5BTitle%5D)+AND+%22Ann+Clin+Lab+Sci%22%5BJournal%5D)">Teratogenic causes of malformations.</a> Ann Clin Lab Sci. 40 (2), 99-114 (2010).</li>
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The authors have nothing to disclose.

The method described here demonstrates the utility of zebrafish in assessing the teratogenic potential of nitrite and nitrate. Compared to other vertebrates, zebrafish have advantages that include high fecundity, external fertilization, optical transparency, and rapid development. Available mutants that lack pigmentation (such as the casper zebrafish36) also help to enhance visibility of internal organs. It is also easy to generate transgenic zebrafish with reporter genes to facilitate analysis in live fish<su...

Teratogenesis is a process that disrupts the normal development of an embryo or fetus by causing permanent structural and functional abnormalities, growth retardation, or miscarriage in severe cases1. It can be caused by certain natural agents (teratogens), which interfere with embryonic development in multiple ways2. During human fetal development, common teratogens such as radiation, infectious agents, toxic metals, and organic chemicals have been reported to cause defects in epicanthic folds (the skin fold in the upper eye lid) and clinodactyly (curved finger or toe) through morphogenetic errors1.
Understanding the molecular mechanism of teratogenesis is the first step towards developing treatment and prevention. Several vertebrate models such as the African clawed frog (Xenopus laevis) and zebrafish (Danio rerio) have been used to determine the molecular pathways affected by teratogens. Previous studies have used zebrafish as a model for epidemiology, toxicology and teratogenesis3-7. Scholz et al. considered zebrafish as a &#34;gold standard&#34; for environmental toxicity assessment. This is due, in part, to the transparency of the zebrafish embryo, which allows researchers to visualize the developmental defect as it occurs8. Approximately 70% of human genes have orthologues in zebrafish, making zebrafish a desirable vertebrate model for studying human defects9.
Some epidemiological studies have suggested that nitrate and nitrite, commonly present in farm foods and water, are associated with birth defects or spontaneous abortions10,11, while other studies do not support this association12. Nitrate (NO3-) and nitrite (NO2-) are naturally present in soil and water. They are a source of nitrogen for plants and are a part of the nitrogen cycle13. Foods such as green beans, carrots, squash, spinach, and beets from farms that use fertilizers high in nitrate have significantly augmented levels of nitrate and nitrite7. Milk from cows fed with high nitrate foods and fish in high nitrate water (mainly from soil runoff30) can lead to humans consuming large amounts of nitrate and nitrite14. Nitrate and nitrite are also commonly used in food preservation, which dramatically increases the amount ingested by humans12.
Optimal levels of nitrate and nitrite play fundamental roles in physiological processes like vascular homeostasis and function, neurotransmission and immunological host defense mechanisms13-15. However, exposure to high levels of nitrate and nitrite may lead to adverse effects, especially in infants and children16. Ingested nitrate is further converted to nitrite in the oral cavity by microflora and in the gastrointestinal tract by intestinal microflora17.
Nitrate puts infants at a high risk for blue baby syndrome by oxidation of hemoglobin to methemoglobin, impairing hemoglobin from its oxygen carrying ability18. This results in the blue color of skin that extends to peripheral tissues in more severe cases. Inhibited oxygenation of tissues results in other symptoms, most severely leading to coma and death19,20. Similar symptoms are observed in babies and adults at higher concentrations of nitrate21. Elevated levels of methemoglobin in adults due to nitrite poisoning results in cyanosis, headache, breathing disorders31, and death if not treated due to complications related to vital tissue hypoxia32,33.
Nitrate ingested at higher levels can also result in various health complications. Childhood diabetes, recurrent diarrhea, and recurrent respiratory tract infections in children have been linked with high nitrate intake11,17,22. Chronic exposure to a high level of nitrate is associated with urination and spleen hemorrhaging. Acute high dose exposure to nitrates can lead to a wide spectrum of medical conditions like abdominal pain, muscle weakness, blood in stools and urine, fainting, and death11. Prenatal exposure to nitrate at high levels has been associated with neural tube and musculoskeletal defect11.
A recent report showed that treating zebrafish embryos with nitrite led to yolk sac edema, craniofacial and axial malformations, and swim bladder noninflation5. In this study, we demonstrate a method for treating zebrafish embryos with nitrate and nitrite to determine their teratogenic potential. Embryos were exposed to nitrite at different concentrations and different lengths of time. Ethanol was used as a positive control, since it is an established teratogen23. Our method showed that both high concentrations and long exposure times to nitrite were detrimental to survival and resulted in various phenotypes, ranging from mild (edema) to severe (gross developmental defects). Therefore, the zebrafish is a useful model for directly exploring the potential teratogenic effects of nitrate and nitrite on embryos to complement epidemiological studies.

<table><tbody><tr><td>DREL/2010 instrument</td><td>Hach</td><td>26700-03</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>KIMAX glass Petri Dish</td><td>VWR</td><td>89001-244</td><td></td></tr><tr><td>MS-222</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>NitraVer 5 Nitrate Reagent</td><td>Hach</td><td>14034-46</td><td></td></tr><tr><td>NitriVer 3 Nitrite Reagent</td><td>Hach</td><td>14065-99</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>3-374-10</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>S6E stereomicroscope</td><td>Leica</td><td>10446294</td><td></td></tr><tr><td>Sodium nitrate</td><td>Fisher Scientific</td><td>S343</td><td></td></tr><tr><td>Sodium nitrite</td><td>Fisher Scientific</td><td>S347</td><td></td></tr><tr><td>Transfer pipets</td><td>Laboratory Products Sales</td><td>L320072</td><td></td></tr><tr><td>Glass vials</td><td>Fisher Scientific</td><td>03-338B</td><td></td></tr></tbody></table>

zebrafish as a model to assess the teratogenic potential of nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to 300 mM ethanol for 22 hr had no effect on survival (data not shown), consistent with previous reports5,23,26. This is expected, as ethanol is a known teratogen and served as a positive control. Observed phenotypes included pericardial edema, swim bladder noninflation (Figure 1), craniofacial defects, and developmental delay (data not shown).
Treatment with nitrite resulted in mild to sever...

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Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

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9.4K 조회수.  Indiana University of Pennsylvania. Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

비디오: Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

연구

의학

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

제브라피시 유충에 대한 환경 오염물질의 신경행동 효과 연구

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

환경과학

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

잠재적인 방사능 감작제 또는 보호제를 평가하기 위한 모델로서의 제브라피시 유충

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

암 연구학

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.
One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).
ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

제브라피쉬에서 백금 및 루테늄 기반 화합물 노출의 선량 흡수는 유도적으로 결합된 플라즈마 질량 분광법과 광범위한 응용 분야에 의한 것입니다.

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the ...

화학

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Zebrafish 배아를 이용한 화학 화합물의 독성에 대한 신속한 평가

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

생물학

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

성인 얼룩말 피시에 있는 Cisplatin에 의해 유도된 급성 신장 상해 모형

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

성인 제브라피쉬의 신경 독성 효과를 평가하기 위한 행동 테스트 배터리

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

저자 스포트라이트: 성체 제브라피시의 환경 및 약물 유발 행동 변화 평가 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

요약

더 많은 비디오 탐색

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

제브라피시 유충에 대한 환경 오염물질의 신경행동 효과 연구

잠재적인 방사능 감작제 또는 보호제를 평가하기 위한 모델로서의 제브라피시 유충

제브라피쉬에서 백금 및 루테늄 기반 화합물 노출의 선량 흡수는 유도적으로 결합된 플라즈마 질량 분광법과 광범위한 응용 분야에 의한 것입니다.

Zebrafish 배아를 이용한 화학 화합물의 독성에 대한 신속한 평가

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

성인 얼룩말 피시에 있는 Cisplatin에 의해 유도된 급성 신장 상해 모형

단일 탱크에서 행동 테스트 배터리를 사용한 성인 Danio rerio 의 신경 독성 평가

단일 탱크에서 행동 테스트 배터리를 사용한 성인 Danio rerio 의 신경 독성 평가

단일 탱크에서 행동 테스트 배터리를 사용한 성인 Danio rerio 의 신경 독성 평가