This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

NOTE: Ethics Statement: All experiments were conducted in accordance with Massachusetts General Hospital guidelines for animal use in research.
1. Advance Preparation
<ol>
	<li>Determine how many adult zebrafish 6-12 months old to injure. Plan to injure 10-20% more fish than will be needed. In order to minimize variability, use age matched sibling fish reared together in the same tank so that they are roughly the same size and have less genetic variability. Female fish are much easier to inject compared to the male fish, due to the larger capacity of the abdomen. However, males and females give similar results.</li>
	<li>When doing the experiment for the first time, plot a dose curve to determine the appropriate gentamicin dosage for a specific strain of fish. Test each new batch of gentamicin before use in experiments since the purity of gentamicin varies from batch to batch. Proper dosage should be determined by assessing expression of injury markers such as lhx1a. (See Figure 1A-D).</li>
	<li>Prepare gentamicin solution. Gentamicin stock solution may be stored in -20 &#176;C and thawed for later use. For example, for an 80 mg/kg dose (80 mg gentamicin/kg of bodyweight) in a fish weighing 0.5 g, prepare 2 mg/ml of gentamicin in phosphate buffered saline as a convenient working solution. This provides a 20 &#181;l of gentamicin for intraperitoneal injection into the fish. 
	NOTE: In general, 80-120 mg/kg achieve good results, but doses as low as 40 mg/kg may be appropriate. Gentamicin may also be purchased in solution to minimize hazardous exposure to lab personnel. 
	NOTE: Gentamicin in high doses can be toxic. Wear gloves and mask when weighing powder.</li>
	<li>Prepare 100 ml of 0.016% tricaine water for anesthetizing fish. Prepare a 25x stock of tricaine (4 g/1 L) dissolved in sterile milliQ water adjust to pH 7 and store at 4 &#176;C until use. Dilute 4 ml in 100 ml of fish water for a working concentration of 0.016%. 
	NOTE: Tricaine is an anesthetic and skin irritant, wear gloves when handling.</li>
	<li>Make a fish scoop out of a plastic transfer pipet by cutting the bulb into a scoop shape and cutting 2 slots in the bottom to drain water. Load 1ml syringes (with 10 &#181;l gradations) with the prepared gentamicin solution and attach a 30&#189; G needle. Remove any air bubbles. Twist the needle on the syringe to make sure that the angled tip on the needle is facing away and the syringe markings are facing forward and readable. For control injections, prepare a syringe and needle with sterile PBS.</li>
	<li>Prepare a scale with a clean surface or weigh boat as well as paper towels for drying fish and for holding fish for injection.</li>
	<li>Prepare individual small half-liter containers with lids for holding fish for observation overnight post injection. Small transparent plastic mating cages are useful for this purpose. These containers should NOT be white - ideally they should be clear so that the white epithelial casts shed by the fish can be observed on a black bench top. Fill each container with enough fish water for the fish to swim comfortably.</li>
</ol>
2. Intraperitoneal Injection of Gentamicin
<ol>
	<li>Anesthetize the fish in 0.016% tricaine solution in fish water. Wait for the gill ventilation rate to slow and for the fish to no longer respond to touch.</li>
	<li>Scoop up the fish using the fish scoop, moving from head toward tail in order to avoid injuring the gills or fins. Place the fish on paper towels to absorb the excess water by turning the fish out gently on its side and shake off excess water from the scoop.</li>
	<li>Scoop up the fish again and place it in the weigh boat on the zeroed scale and weigh the fish. Round to the nearest 0.25 g, and calculate the appropriate amount of gentamicin to inject. 
	NOTE: For a 0.5 g fish use a 20 &#181;l injection at 80 mg/kg dose.</li>
	<li>Scoop up the fish again and place it on a dry folded paper towel. If injecting using the right hand, hold the paper towel in the left hand and place the fish&#8217;s head pointing left, with the belly easily accessible.</li>
	<li>Hold the syringe with the needle at a 45&#176; angle to the skin of the belly, anterior to the cloaca. Push the needle just under the skin, then decrease the angle and slide the needle forward under the skin, avoiding the internal organs. Depress the plunger the appropriate amount, pause to make sure no liquid is coming out around the needle, then withdraw the needle. If holding the fish steady is a problem, brace elbows against torso to stabilize them. 
	NOTE: Wear gloves when handling gentamicin solution.</li>
	<li>Drop each fish into an individual container. Observe the fish and ensure its recovery from anesthesia. Keep fish at 28.5 &#176;C overnight. 
	NOTE: If the fish doesn&#8217;t revive immediately, use a plastic transfer pipet to irrigate water across its gills to revive it.</li>
	<li>Use the same syringe and needle when injecting multiple fish with the same dose of gentamicin. Dispose of the used syringe and needle into the appropriate biohazard sharps container.</li>
</ol>
3. Post Injection Observation of Injury
<ol>
	<li>The next day (1 day post injury), place the injected fish in its container on a dark surface. White casts of dead epithelial tissue excreted by the injured fish should be visible. (See Figure 1E-H). If there are no casts, either the fish was not injured by the gentamicin injection (either the dose was too low, or some of the gentamicin leaked during the injection process) or the fish was severely injured resulting in complete blockage of the ureters and cloaca with sloughed tissue. 
	NOTE: If the fish is unable to clear the casts from its body it will usually die within 2-3 days and is therefore unusable for longer assays. If no casts are visible, euthanize the fish in tricaine water by immersing for at least 10 min after gill movement stops.</li>
	<li>If white tissue casts are visible, set the fish aside and continue checking the other fish. An appropriate dose of gentamicin will result in 80-90% of the fish being usable. Pool the injured fish and then split them into different treatment groups if desired.</li>
</ol>
4. Care of Recovering Fish
<ol>
	<li>Keep fish in clean water and uncrowded environment. Dirty water and crowding will lead to infections and unintended death. Try to keep no more than 6 fish/500 ml of fish water and change the water daily if possible. 
	NOTE: This is a minimum volume for conservation of space or if the investigator wishes to perform experiments using expensive drug treatments.</li>
	<li>Do not feed the fish until 3 days post injury. Recovering fish will not eat at first, and any food in the tank will decompose and promote bacteria growth. Starting at 3 days post injury, feed a small amount once a day.</li>
</ol>
5. Analysis of Injured Kidneys
<ol>
	<li>For in situ hybridization for lhx1a mRNA expression, kidneys can be harvested from fish at desired timepoints. Euthanize fish in tricaine water on ice at least 10 min until gill movement stops, then remove the head with a razor blade and open the body cavity and remove the internal organs with forceps.</li>
	<li>Leave the kidney in place (pigmented organ attached to the dorsal body wall) and fix the fish in 4% paraformaldehyde in PBS overnight. Dissect out the kidney15 and continue with standard in situ hybridization.
	<ol>
		<li>Briefly, wash the kidneys in PBST (phosphate buffered saline 0.5%Tween 20), permeabilized with proteinase K in PBST (10 &#181;g/ml) for 1 hr at room temperature. Post-fix with 4% paraformaldehyde, and washed again with PBST.</li>
		<li>Then pre-hybridize the kidneys overnight at 68&#160;&#176;C&#160; in hybridization buffer (50% formamide, 5x SSC, 50 &#181;g/ml heparin, 500 &#181;g/ml tRNA, 0.1% Tween20, pH 6.0).</li>
		<li>Incubate the samples with digoxigenin labeled probe in hybridization buffer, then wash for 5-10 min each with 100%, 75%, 50%, 25% hybridization buffer at 68&#160;&#176;C, then moved to room temperature and wash twice for 30 min with 2x SSC with 0.1% Tween20, and wash twice for 30 min with 0.2x SSC with 0.1% Tween20.</li>
		<li>Equilibrate the sample 3x 5 min in MAB (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) and blocked overnight at 4 &#176;C in MAB with 10% goat serum. Then incubate samples overnight in anti-digoxigenin-AP Fab 1:5,000 in MAB.</li>
		<li>Wash the sample 5x for 1 hr in MAB, and equilibrate 3x for 15 min in NTMT (0.1 M Tris pH9.5, 0.05 M MgCl2, 0.01 M NaCl, 50 &#181;l Tween20).</li>
		<li>Then treat the kidneys with NBT/BCIP to detect signal. Fix the kidneys with 4% paraformaldehyde and incubated in dimethylformamide to remove excess NBT/BCIP, bleached overnight in deionized water. Wash the kidneys and then photograph in PBST with 50% glycerol under a coverslip.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have no competing financial interests to disclose.

The zebrafish is ideal for studying regeneration of adult organs including the adult mesonephric kidney6. Recent studies have taken advantage of molecular markers and new transgenic reporter lines to better characterize the steps that occur during nephron regeneration and what cells might be responsible7,12. Observation of urinary casts has been used for over a century to diagnose kidney disease in humans16. Here we use the presence of casts as an easy, noninvasive and early indicator of ...

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. Although mammals are able to repair existing nephrons after injury using differentiated epithelial cells1-4, they seem to lack a pool of reserved stem cells5 and are unable to form new nephrons de novo. In contrast to mammals, adult fish are able to form new nephrons throughout adult life to support the growth of the fish and in response to injury6,7. The zebrafish, Danio rerio, is an invaluable model organism for the study of organ regeneration8-10 and has the potential to provide powerful insights into applications for engineering the repair of human kidneys. The Tg(Lhx1a:EGFP) transgene11 has been shown to label a pool of nephron progenitor cells in the adult zebrafish kidney12, however mechanisms controlling the response of lhx1a+ cells to injury remain unclear.
The aminoglycoside gentamicin is a widely used antibiotic with known nephrotoxic and ototoxic effects in humans13. Intraperitoneal injection of gentamicin is an established method of inducing acute kidney injury in fish6. This injury in fish mimics the loss of tubular epithelium and scarring of glomeruli that occurs in humans after gentamicin overdose14. Inducing injury in zebrafish by gentamicin injection is a convenient way of inducing a strong, synchronous regeneration response, with many new nephrons produced and simultaneously proceeding through stages of formation, proliferation and differentiation.
This protocol details our method for robust and reproducible injury in the adult zebrafish kidney by utilizing a noninvasive visual screening process to minimize outliers. We take advantage of the fact that injury with gentamicin leads to death of epithelial kidney tissue and formation of renal tubular casts, which then accumulate into masses in the mesonephric ducts and cloaca. These are passed by the fish and can be observed visually in the water. This allows us to screen for fish with strong nonlethal injury, which can then be pooled for further experimentation. Minimizing the numbers of uninjured fish or fish that die before they reach the endpoint of the experiment leads to more uniform and efficient data collection and analysis. In addition, no special devices or reagents are required, making this method cost-effective and appropriate for use in an academic or teaching setting. Using our method, here we show the increasing effects of gentamicin dose on kidney regeneration as well as the effect of increased temperature.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin sodium sulfate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">G1264</td>
			<td>TOXIC, purity varies from batch to batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic transfer pipets</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 ml Norm-Ject syringes</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">72520</td>
			<td>green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30G1/2 needles</td>
			<td style="width:101px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>IRRITANT</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">16% paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td>Make 4% in 1xPBS for working solution</td>
		</tr>
	</tbody>
</table>

kidney regeneration in adult zebrafish by gentamicin induced injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Gentamicin injury can be confirmed visually by the observation of renal epithelial casts in the water (Figure 1). Different dosages of gentamicin were used to injure adult wildtype TuAB zebrafish, resulting in increasing numbers of lhx1a+ cellular aggregates in the regenerating kidney (Figure 1A-D). White casts can easily be seen in the water 1 day after injury (Figure 1E-H). A low dose of gentamicin resulted in most fish having...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Research

JoVE Journal

Developmental Biology

10.7K Views.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 

Video: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...