The authors have nothing to disclose.

Toutes les procédures sont effectuées suivant les directives décrites dans les «Principes de protection des animaux de laboratoire» (National Institutes of Health publication no. 85-23, révisée 1985) et le protocole de l&#39;animal Comité institutionnel de protection et d&#39;utilisation des animaux Université Rosalind Franklin approuvé 08-19. 1. Gentamicine-induction de cheveux nécrose des cellules <ol><li> Préparer le sulfate de gentamicine dans du sérum physiologique à une concentration finale de 0,004% (4,32 mM). </li><li> Placer le poisson adulte (D. rerio, 4-6 mois) dans un récipient contenant le 0,004% (4,32 mM) de solution de gentamicine. Tout récipient peut être utilisé, mais nous utilisons un conteneur de poissons à partir d&#39;un système aquatique Pharmacal qui est 7 de large, 6 en haut, et 7 dans longtemps. Placer le récipient avec des poissons dans un incubateur réglé à 28 ° C pendant 24 heures. Régler le volume total de fluide dans le réservoir à un niveau suffisant pour maintenir le poisson dans un état viable pour la période de 24 heures. Remarque: L&#39;aération du fluide gentamicine n&#39;est pas necessaire si suffisamment de volume est utilisée pour le nombre de poissons à traiter. </li></ol> 2. Coloration vitale des cellules ciliées <ol><li> Préparer une concentration de 0,08% (dans une solution saline normale) du colorant vital fluorescent [4-4-diethylaminostyryl)-N-méthylpyridinium (485 nm excitation λ et 603 nm émission λ dans le methanol) à partir d&#39;une solution de travail de réserve de 15 mg / ml dans de l&#39;éthanol. </li><li> Pour déterminer si le traitement a été efficace gentamicine un sous-ensemble de contrôle et de la gentamicine poissons traités sont colorées immédiatement en plaçant le poisson dans le puits d&#39;une plaque de culture 6 puits contenant le colorant vital. Utilisez un nombre suffisant de poissons (et des plaques de culture comme l&#39;exige la signification statistique à atteindre. Basé sur la vitesse de l&#39;examinateur de comptage neuromastes, placer le poisson dans les plaques de manière échelonnée dans le temps afin que les poissons ne sont pas tachés de plus de 75 min comme décrit dans l&#39;étape 2.3. </li><li> Placez les plaques de l&#39;étape 2.2 dans un tiroir de banc par le microscope à fluorescence àêtre utilisé pour l&#39;examen de neuromastes colorées. Éteignez les lumières de la pièce pour éviter l&#39;extinction du colorant vital au cours de la période de coloration 1 h à température ambiante. </li><li> Préparer la fois colorant wash-out et des réservoirs d&#39;eau anesthésiques. Eau de wash-out colorant est de l&#39;eau de poisson normale et de l&#39;eau anesthésie, ajouter suffisamment de 2-phénoxyéthanol sorte qu&#39;une dilution de 1:1000 dans l&#39;eau du poisson normale est atteint. </li><li> Placer le poisson dans l&#39;eau en excès de poisson normale pour rincer l&#39;excès de colorant vital et passez à l&#39;étape 3.1 pour l&#39;observation de colorant teinté poissons vital. </li><li> Pour la régénération de la neuromastes, transférer les poissons de la gentamicine traités qui ont été lavés à l&#39;eau du poisson normale dans un incubateur pour entre 8-16 heures à 28 ° C. </li><li> À divers moments entre 8-16 heures, les poissons sont retirées de l&#39;incubateur, lavées et colorées comme indiqué dans les étapes 2.1 à 2.4. Passez à l&#39;étape 3.1 pour l&#39;observation de colorant teinté poissons vital. </li></ol> 3. Anesthésier le poisson et comptage fluorescent de Neuromastes <ol><li&gt; Blot chaque poisson sur une serviette en papier pour enlever l&#39;excès de liquide, puis placez-le sur un morceau de papier humide de filtre qui est centrée sur le couvercle d&#39;une boîte de Petri en plastique. </li><li> Placer le couvercle sur la scène d&#39;un microscope stéréo fluorescent pour obtenir une image numérique des colorants neuromastes colorées vitaux des points du corps milieu. </li><li> Utilisez un appareil photo numérique placé sur le microscope stéréo fluorescent fixé un grossissement de 2X pour capturer des images pour l&#39;analyse quantitative ultérieure. Remarque: Le réglage de grossissement du microscope stéréo peut dépendre de la marque de microscope utilisé, mais le réglage doit permettre l&#39;affichage et le comptage des neuromastes individuels facile dans le milieu des points du corps. </li><li> Déterminer la quantité de régénération en comptant le nombre de neuromastes visibles dans les quatre points désignés sur le côté inférieur le plus ventrale du poisson juste en amont de la nageoire pectorale droite (voir la figure 1). Pour l&#39;analyse statistique en utilisant un test approprié tel que ANOVA or T-test de Student. Les expériences doivent utiliser un minimum de 5 poissons par point de temps et toutes les expériences doivent être répétées au moins 3x. </li><li> Sur la base de la courbe de temps neuromastes de régénération (voir la figure 3), compter neuromastes entre 8-16 heures après la gentamicine wash-out pour être dans la phase linéaire de la courbe de régénération. Remarque: L&#39;utilisation de la phase de temps linéaire permet une analyse quantitative adéquate entre les groupes témoins et expérimentaux. </li></ol> 4. Comptage fluorescent de cellules ciliées individuelle pour l&#39;obtention d&#39;une résolution plus élevée de l&#39;analyse quantitative si l&#39;analyse neuromastes n&#39;est pas statistiquement significative <ol><li> Si l&#39;analyse quantitative au niveau de neuromastes n&#39;est pas significative, une analyse au niveau de la cellule individuelle de cheveux peut également être utilisée pour obtenir un plus haut degré de résolution. Sélectionnez poisson à un particulier le point de temps après la gentamicine de wash-out (point de temps sur la base des études de neuromasts antérieures), colorant vital stain le poisson tel que décrit dans le protocole 2, puis euthanasier les poissons en utilisant le 2-phénoxyéthanol à une dilution 1:500 1-5 min. </li><li> Dans une lumière tamisée pour éviter l&#39;extinction, faire quatre incisions ainsi qu&#39;un carré préparation de lambeau de peau est faite comme suit. Faire une incision le long des côtes supérieures du poisson jusqu&#39;à ce qu&#39;il soit aligné avec les nageoires anales, puis faire une incision dans le ventre, et enfin, faire deux incisions verticales de chaque côté de ces incisions de sorte que le volet carré de la peau est créé. Note: Cette préparation de la peau intégrera les points du corps milieu utilisés dans les expériences de neuromasts. </li><li> Placer l&#39;échantillon de peau sur une lame de verre, puis placez une circulaire lamelle de verre sur le spécimen excisé de la peau pour aider à l&#39;ancre et aplatir le tissu pour l&#39;imagerie numérique ultérieure. </li><li> En utilisant les échantillons de peau provenant de l&#39;étape 4.3, d&#39;obtenir des images numériques de cellules ciliées dans chaque neuromastes des points milieu du corps. Prenez des photos à un grossissement d&#39;au moins 60X et puis de compter le CE de cheveuxlls dans neuromastes individuels pour l&#39;analyse quantitative comparative de la commande et les groupes expérimentaux (voir figure 4). </li></ol>

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This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

Basé sur le vaste corpus de littérature qui a été mis en place pour l&#39;analyse de la ligne latérale (LL) de la régénération chez le poisson zèbre embryonnaire et larvaire 8,24,25, le but de notre étude était de développer un dosage quantitatif pour la régénération de la ligne latérale chez le poisson zèbre qui pourrait être appliquée à des modèles de maladies qui sont les mieux étudiés dans les poissons adultes. Nous avons constaté que certains points critiques sont importantes lors...

Le système (LL) de la ligne latérale est un organe mécanosensorielle trouvé dans les deux poissons et les amphibiens qui est responsable de l&#39;audition, l&#39;équilibre, rhéotactisme et les comportements de médiation telles que la scolarisation et l&#39;évitement des prédateurs 1-5. Elle est composée de grappes de cellules ciliées entourées par des cellules de soutien, qui sont tous deux positionnés dans des structures appelées neuromastes 6. Ces neuromastes sont généralement organisés en lignes verticales (appelé points) le long de l&#39;axe longitudinal du corps et de la queue avec quelques points horizontaux observés dans la tête du poisson. Chez l&#39;adulte, neuromastes sont beaucoup plus nombreux dans les points par rapport à l&#39;embryon ou larves de poissons 6. Études biomédicales chez le poisson zèbre ont porté sur l&#39;effet du traitement antibiotique, traumatisme induit par le bruit, une infection chronique, etc. sur les cellules ciliées 7,8 pour tenter de mieux comprendre leurs effets sur les humains. Contrairement à la plupart des vertébrés, teleosts, tels que le poisson zèbre (Danio rerio), ont la capacité de régénérer les cellules de cheveux perdus. Poisson zèbre sont particulièrement utiles en raison de leur temps de développement rapide et de haute capacité de régénération. A ce jour, toutefois, études de poisson zèbre sur le développement et / ou la régénération de la ligne latérale ont principalement utilisé le poisson embryonnaire et larvaire stade en raison de la réduction du nombre de neuromastes de la ligne latérale qui permet de faciliter le comptage et l&#39;analyse 6,9,10. Cependant, comme de nombreux modèles de poisson zèbre de maladies neurologiques et non neurologiques 11-16 sont étudiés dans les poissons adultes et non les larves, nous nous sommes concentrés sur le développement d&#39;un test de régénération de la ligne latérale chez le poisson zèbre adulte en utilisant la gentamicine (un aminoglycoside déjà utilisé dans les larves de poisson zèbre et plus récemment utilisé avec les poissons adultes 17) de sorte qu&#39;une analyse était disponible qui pourraient être appliquées à des modèles de maladies adultes actuels de poisson zèbre. Bien que les procédures précédemment publié par Van Trump et al. 17 fixe les conditions pour l&#39;ablation des cellules ciliées dans les poissons adultes, ils n&#39;ont pas établi une courbe standard pour la régénération neuromastes qui est nécessaire pour la comparaison quantitative entre les groupes témoins et expérimentaux tels que l&#39;utilisation de lignes de poisson zèbre transgénique ou états pathologiques pharmacologiquement induits chez le poisson zèbre 18. Nous avons donc suivi les procédures de Van Trump et al. 17 pour l&#39;ablation des cellules ciliées, mais construit sur ​​leur travail pour établir une courbe standard de régénération neuromastes pour permettre aux enquêteurs d&#39;utiliser nos données lorsque l&#39;on compare les groupes témoins et expérimentaux tels que des modèles de la maladie de poisson zèbre adulte . L&#39;essai a également été conçu pour permettre l&#39;extension de l&#39;analyse à la cellule individuelle lorsque les cheveux un plus haut niveau de résolution est requise.

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

En raison de l&#39;importance clinique de l&#39;ouïe et troubles de l&#39;équilibre chez l&#39;homme, des organismes modèles tels que le poisson zèbre ont été utilisés pour étudier le développement de la ligne latérale et la régénération. Le poisson zèbre est particulièrement intéressante pour ces études en raison de son temps de développement rapide et sa grande capacité de régénération. À ce jour, les études de poisson zèbre de latéral régénération de ligne ont principalement utilisé des poissons des stades embryonnaires et larvaires en raison de la diminution du nombre de neuromastes à ces stades. Cela a fait de l&#39;analyse quantitative de la ligne de régénération / et ou le développement plus facile dans les premiers stades de développement latéral. Parce que de nombreux modèles de poisson zèbre de maladies neurologiques et non neurologiques sont étudiés dans les poissons adultes et non dans l&#39;embryon / larves, nous nous sommes concentrés sur le développement d&#39;un test latéral quantitative ligne de régénération chez le poisson zèbre adulte ainsi qu&#39;une analyse était disponible qui pourrait être appliquée à courant modèles de la maladie de poisson zèbre adulte. S&#39;appuyant sur des études précédentes par Van Trump et al 17. qui décrit les procédures d&#39;ablation de cellules ciliées de poissons adultes mexicain aveugle de la grotte et le poisson zèbre (Danio rerio), notre test a été conçu pour permettre une comparaison quantitative entre les groupes témoins et expérimentaux. Cela a été réalisé en mettant au point une courbe étalon de neuromastes régénérative sur la base de la réapparition de neuromastes pour cent pendant une période de temps de 24 heures suivant la nécrose induite par la gentamicine, de cellules ciliées dans une région définie de la ligne latérale. L&#39;essai a également été conçu pour permettre l&#39;extension de l&#39;analyse au niveau des cellules ciliées individu quand un niveau supérieur de résolution est requise.

Optimisation des procédures pour quantifier neuromastes régénération de la ligne latérale chez le poisson zèbre adulte. Les neuromastes de poisson zèbre larves sont facilement quantifiables; Cependant, la ligne latérale de poisson zèbre adulte a un plus grand nombre de neuromastes par point faisant des analyses quantitatives plus difficile 6,17,19,20. Comme on le voit sur ​​la figure 1A, la tête a un nombre significativement plus él...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

Parce que de nombreux modèles de poisson zèbre de maladies neurologiques et non neurologiques sont étudiés dans les poissons adultes plutôt que l&#39;embryon / larve, nous avons développé un dosage latéral de régénération de ligne quantitative qui peut être appliquée à des modèles de maladies de poisson zèbre adulte. Le dosage impliqué résolution au neuromastes 1) et 2) les niveaux cellulaires de cheveu.

Un test pour ligne latérale de régénération à l&#39;âge adulte de poisson zèbre

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K vues.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  Parce que de nombreux modèles de poisson zèbre de maladies neurologiques et non neurologiques sont étudiés dans les poissons adultes plutôt que l'embryon / larve, nous avons développé un dosage latéral de régénération de ligne quantitative qui peut être appliquée à des modèles de maladies de poisson zèbre adulte. Le dosage impliqué résolution au neuromastes 1) et 2) les niveaux cellulaires de cheveu. 

Vidéo: An Assay for Lateral Line Regeneration in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

La transplantation de moelle rein entier chez le poisson zèbre pour adultes

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Recherche

Biologie

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Toxicité cellulaire des Nanogenomedicine dans la lignée cellulaire MCF-7: test MTT

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Rétro-orbitaire d&#39;injection chez le poisson zèbre pour adultes

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Injection intrapéritonéale en Zebrafish adultes

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En électroporation in vivo de morpholinos dans la régénération des adultes Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Collecte de sang pour analyse biochimique chez le poisson zèbre adulte

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavage poisson zèbre pour adultes

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Collecte de sang répétées pour des tests sanguins dans le poisson zèbre adulte

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

Électrocardiographie de surface in Vivo pour le poisson zèbre adulte

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...