This work was supported in part by the NIH grant DP2OD008470. Additionally, RAM was supported in part by funds provided by the University of Notre Dame Graduate School. We thank the staffs of the Department of Biological Sciences, the Center for Zebrafish Research, and the Center for Stem Cells and Regenerative Medicine at the University of Notre Dame. We especially thank the members of lab for engaging discussions about kidney biology and their helpful feedback on this work.

The procedures for working with zebrafish embryos described in this protocol were approved by the Institutional Animal Care and Use Committee at the University of Notre Dame.
1. Preparation of Solutions
<ol>
	<li>Make a 50x stock solution of E3 embryo media by mixing 73.0 g NaCl, 3.15 g KCl, 9.15 g CaCl2, and 9.95 g MgSO4 in 5 L of distilled water, and store at RT.</li>
	<li>For culturing of zebrafish embryos, dilute the 50x stock solution of E3 embryo media stock to a 1x working solution with distilled water, then add 200 &#956;l of 0.05% methylene blue to every 1 L of 1x E3 to act as a fungicide, and store at RT.</li>
	<li>Make a pigmentation blocking solution of E3 embryo media with 0.003% 1-phenyl-2-thiourea (PTU) by combining 40 ml of the 50x E3 stock with 0.06 mg PTU. Then bring the volume to a total of 2 L with distilled water.</li>
	<li>Stir the E3/PTU O/N at RT to get the PTU powder into solution. Then store the E3/PTU at RT. 
	Note: The E3/PTU has a shelf life of approximately one week, and older solutions will be less effective at blocking pigmentation development in zebrafish larvae.</li>
	<li>Make an anesthetic solution of 0.2% Tricaine (MS-222) by adding 1 g of Tricaine to 500 ml of distilled water and adjust the pH to 7.2 with 1 M Tris, pH 9.5.</li>
	<li>Prepare to make a 2% methylcellulose solution for imaging by placing a solution of 1x E3 (no methylene blue), with 1/10th volume of 0.2% Tricaine added at 4 &#176;C. 
	Note: The methylcellulose solution is a thick, clear solution that is used to gently stabilize live embryos for microscopy. After manipulations are performed the embryos can be washed in 1x E3 to dissolve the methylcellulose without injuring the animal and enabling microscopy at subsequent stages in the same specimen.</li>
	<li>When cooled to 4 &#176;C, stir the 1x E3/Tricaine solution vigorously on a stir plate and gradually add the appropriate amount of methylcellulose powder while continuing to stir vigorously. Gradually add the powder to the solution to prevent the formation of insoluble methylcellulose aggregates.</li>
	<li>After all the powder is mixed into the 2% methylcellulose/E3/tricaine solution, stir the composite solution in the 4 &#176;C cold room O/N. 
	Note: The cold temperature is best for fully dissolving the powder. If the solution is not clear after one night, stir for an additional workday and/or a second O/N.</li>
	<li>To remove air bubbles from the 2% methylcellulose/E3/tricaine solution, aliquot the mixture into 1.5 ml tubes and spin at high speed (12,000 x g) at 4 &#176;C for 30 min or up to 2 hr.</li>
	<li>Place the 2% methylcellulose/E3/Tricaine solution at 4 &#176;C for long-term storage. 
	Note: Prior to usage, aliquots should be pre-warmed to RT for working with the zebrafish specimens.</li>
</ol>
2. Preparation of Tools
<ol>
	<li>Prepare an embryo manipulator tool, used for positioning embryos for microinjection, by attaching a gel loading tip on the end of a 5&#34; straight dissecting needle, and secure with tape or superglue.</li>
	<li>Prepare microinjection needles using a needle puller, by first placing a fire polished 10 cm borosilicate glass with filament into the instrument and secure the borosilicate glass by tightening the handles.</li>
	<li>Pull the borosilicate glass to fashion fine-tapered needles. Use the following settings: heat 540, pull 245, velocity 200, and time 125.</li>
	<li>Place needles inside a Petri dish, suspending them on a strip of modeling clay to protect the needle tips, and keep the dish covered to prevent dust accumulation.</li>
	<li>To finish preparing the needle for microinjections, use a razor blade or fine forceps edge to cut the pulled end of the needle to create a sharp angular injection tip with a diameter of approximately 0.05-0.1 mm.</li>
	<li>To make the microinjection tray, prepare a 1.5% agarose/E3 solution in a 100 ml Erlenmeyer flask and dissolve the agarose by heating the E3 to a boil in a microwave then allow to cool at RT for approximately 10 min.</li>
	<li>Pour the cooled agarose/E3 solution into a Petri dish to make a foundation with a depth of approximately 0.5 cm. When this has solidified pour a second layer with a depth of approximately 0.3 cm and insert the prefabricated embryo well mold and allow the agarose to solidify at RT. 
	Note: When inserting the mold into the top agar/E3 layer, placing the mold into the agar at an angle to decrease the number of air bubbles.</li>
	<li>When the entire microinjection tray has set, lift the prefabricated embryo well mold out slowly and carefully using a scoopula, then fill the dish with E3 solution and store at 4 &#176;C.</li>
</ol>
3. Embryo Preparation
<ol>
	<li>Set up zebrafish mating tanks by placing dividers into mating chambers and fill with system water.</li>
	<li>Place one fish on each side of the divider such that each mating chamber contains one female and one male fish, and cover each mating chamber.</li>
	<li>The next morning, remove the dividers to enable spawning.</li>
	<li>Check the fish after approximately 30 min, and after returning the adults to the aquarium, collect their embryos by passing the mating tank water through a fine wire-mesh strainer tool.</li>
	<li>Invert the strainer over a Petri dish and rinse with E3 to collect the embryos.</li>
	<li>Incubate the embryos at 28.5 &#176;C O/N.</li>
	<li>When embryos have reached the 24-26 somite stage26, decant most of the E3 media and then dechorionate by adding 100 &#181;l of 50 mg/ml pronase to each dish of embryos and incubating the dish at RT (23 &#176;C) for approximately 15 min. 
	Note: It will take approximately 24-25 hr from the time of fertilization for the embryos to reach the 24-26 somite stage if they are collected in the manner described and incubated O/N at 28.5 &#176;C.</li>
	<li>Fill the dish with E3 and then swirl gently to dislodge the chorions from the embryos.</li>
	<li>Decant the E3/pronase and rinse the dish with 25 ml of fresh E3. Repeat rinse step to remove all pronase.</li>
	<li>To block the development of pigmentation, decant the E3 and replace with 25 ml of fresh E3/PTU when the embryos have reached 24 hr post fertilization. 
	Note: For experiments spanning several days, replace the E3/PTU solution with fresh media once daily to keep the dish clean.</li>
</ol>
4. Microinjection of Nephrotoxin Solution
<ol>
	<li>On the day of injection, prepare the desired nephrotoxin solution along with the appropriate vehicle control.</li>
	<li>Vortex or mix gently to insure the drug(s) is/are in solution, checking the sides of the tube and top of the water column. 
	Note: Nephrotoxin solutions can be prepared to contain trace amounts of fluorescently conjugated dextran to monitor microinjection efficiency and also assess renal clearance at subsequent time points20,28.</li>
	<li>Load a trimmed microinjection needle with ~2-3 &#181;l of the nephrotoxin by threading a fine gel loading tip into the back of the needle and suspend the needle vertically with a piece of tape to allow the solution to fill the trimmed needle tip by gravity.</li>
	<li>Once the needle tip is full, secure the loaded needle in the micromanipulator.</li>
	<li>Test the microinjection volume by placing a drop of mineral oil onto a micrometer slide and inject into the oil to evaluate the droplet size. An injection volume of 500 pl has a diameter of 0.1 mm.</li>
	<li>Remove the dish of embryos from the incubator, and anesthetize by adding approximately 5 ml of 0.2% Tricaine to the embryo dish.</li>
	<li>To ensure complete anesthetization, gently touch embryo with the tip of the embryo manipulator tool. Lack of movement indicates sufficient anesthetization.</li>
	<li>After successful anesthetization, transfer embryos to the injection mold with a transfer pipette.</li>
	<li>Maneuver each embryo into a different well of the mold placing the head in the deepest area of the well such that the trunk rests along the depression and the tail sticks up out of the depression.</li>
	<li>Insert the filled needle into the micromanipulator and position the needle tip next to an embryo.</li>
	<li>Gently insert the needle into the tail vessel by moving the joystick forward and depress the foot pedal to deliver the microinjection. 
	Note: Successful injection can be gauged by watching the liquid enter circulation. Further, co-injection of the nephrotoxicant with fluorescently conjugated dextran enables the researcher to verify successful injection subsequent to the procedure.</li>
	<li>Gently pull back on the joystick to remove the needle from the embryo.</li>
	<li>After injection, transfer the embryos to a clean dish and rinse to remove the Tricaine and replace with fresh E3/PTU.</li>
	<li>Incubate the embryo to the desired time point to assess morphology, which can be documented by photography in methylcellulose mounting media, or process for experimental analysis as desired. 
	Note: Recovery of embryos should be assessed to determine the percentage of individuals with edema, which commonly indicates renal injury.</li>
</ol>

The authors have nothing to disclose.

A diverse number of therapeutic agents have been associated with AKI29. There have been significant research advances in understanding the damage induced by many individual compounds, such as the aminoglycoside gentamicin30 and the widely used chemotherapeutic cisplatin31,32. Some pathological changes involved in these conditions, however, remain the subject of ongoing study. One emergent challenge remains understanding how multiple drugs adversely affect patients, especially those in hig...

AKI is an abrupt loss of kidney function that can lead to devastating health consequences1. AKI is a significant healthcare issue worldwide due to its high incidence of approximately 20% among hospitalized patients, with even higher rates of 30-50% in critical care cases and the elderly, and mortality rates of 50-70%1-3. Unfortunately, the prevalence of AKI has been increasing and is projected to escalate further over the next decade, due in part to the diversity of factors that can induce AKI, which include post-operative stress, ischemia, and exposure to nephrotoxins such as antibiotics and chemotherapeutic drugs4.
AKI involves sudden cellular damage within the kidney, commonly occurring in nephrons, which are the essential functional units, and are comprised of a blood filter and a segmented tubule that drains urine into central collecting ducts1. When a significant number of nephrons are damaged during AKI, the immediate effects include an interruption in waste clearance from the circulation, and reduced or abrogated fluid flow through nephrons due to obstruction from dead and dying cells1. Over time, tubular obstruction can lead to degeneration of entire nephrons, which permanently reduces renal function1. Physiological alterations in the kidney following AKI also involve complex inflammatory events that can lead to chronic scarring1.
Despite these outcomes, nephrons have some capacity to undergo regeneration after AKI that reconstitutes the tubular epithelium5,6. While there has been an increasing molecular understanding of nephron regeneration, the mechanisms remain elusive in many regards and necessitate continued investigation7. The degree to which AKI results in permanent renal damage also remains unknown. Current research suggests the regenerative potential for the kidney is the highest following less severe cases of AKI, while more pronounced or repeated episodes lead to chronic kidney disease (CKD) and culminate in end stage renal disease (ESRD) that requires life-saving transplantation or dialysis8,9. Additionally, individuals already suffering from CKD are at an even higher risk of contracting a severe episode of AKI8,9. Taken together, it is clear that continued basic and clinical research is vital to understand, treat and prevent AKI.
Research with animal models has been instrumental in appreciating the progression of local and environmental alterations that occur during AKI10. To expand this understanding as well as develop new therapies, the zebrafish animal model has been employed in a variety of ways11,12. The nephrons of the zebrafish kidney, in both the embryo and adult, display a high degree of conservation with mammals13-16. Further, nephron epithelial injury in zebrafish resembles the process in higher vertebrates, whereby the local destruction of tubular cells is followed by intratubular proliferation and reestablishment of nephron architecture17-19. In the embryo, however, extensive tubule damage from the nephrotoxins like cisplatin is associated with lethality20,21. By comparison, zebrafish adults survive AKI and exhibit substantive regenerative capabilities in the kidney. For example, following exposure to the aminoglycoside antibiotic gentamicin, zebrafish regenerate tubule epithelial damage and grow new nephron units as well22-24. While these gentamicin-induced AKI studies have provided invaluable information, understanding renal damage from diverse nephrotoxins remains critical to appreciate the effects and response to different types of damage25.
The zebrafish embryo, due to its size, transparency, and genetic tractability, has many benefits for nephrotoxin studies25, where the method of microinjection20,21 is used to administer the molecule(s) for investigation. Nephrons are formed by 24 hr post fertilization (hpf) and begin to filter blood by approximately 48 hpf26,27. Thus, the rapid formation and function of the embryonic kidney facilitates experimental analysis. However, the process of microinjection has technical challenges and there can be a steep learning curve to mastering the technique. In this video article, we describe how to perform microinjections and provide troubleshooting tips in order to enhance the rate of successful injections.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Chloride</td>
			<td style="width:229px;">American Bioanalytical</td>
			<td style="width:129px;">AB01915</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB01652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB00366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td>Aldrich Chemistry</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate (Tricaine)</td>
			<td>Fluka Analytical</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass</td>
			<td>Sutter Instruments Co.</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>Mo. P097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Diposable Petri Dishes, Sterile, Corning:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm x 15mm</td>
			<td>VWR</td>
			<td>25373-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm x 15mm</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;(microinjection tray) 150mm x 15mm</td>
			<td>VWR</td>
			<td>25373-187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Temperature Incubator</td>
			<td>Fischer Scientific</td>
			<td>11 690 516DQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Tweezer</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-5010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Micrometer</td>
			<td>Ted Pella, Inc.</td>
			<td>2280-24</td>
			<td></td>
		</tr>
	</tbody>
</table>

nephrotoxin microinjection in zebrafish to model acute kidney injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

A microinjection station set up includes a stereomicroscope, micromanipulator and pressure regulator (Figure 1A). Transillumination of the injection plate is preferable to view specimens during this procedure (Figure 1B). Preparation of the injection needle involves pulling the appropriate borosilicate glass, followed by preparing the edge with cutting and finally back-loading the needle. Optimally, the needle tip is beveled rather than blunt (Fig...

Watch this Scientific Journal Video about Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury at JoVE.com

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

nephrotoxin-microinjection-in-zebrafish-to-model-acute-kidney-injury

Research

JoVE Journal

Biology

8.8K Views.  University of Notre Dame. Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Video: Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

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.

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

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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.

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

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...