Name: histological analyses of acute alcoholic liver injury in zebrafish
Uploaded: 2017-05-25T13:00:00
Description: Watch this Scientific Journal Video about Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish at JoVE.com

The authors would like to acknowledge Dr. Katy Murray at the Zebrafish International Resource Center; Dr. Stacey Huppert and Kari Huppert at CCHMC, for their helpful advice on the protocol; and CCHMC veterinary service, for the fish care. This work was supported by NIH grant R00AA020514 and a research grant from the Center for Pediatric Genomics at CCHMC (to C.Y.). It was also support in part by NIH grant P30 DK078392 (Integrative Morphology Core) of the Digestive Disease Research Core Center in Cincinnati.

AB WT adult and larval zebrafish were maintained under standard conditions21 in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1985); their use was approved by the Institutional Animal Care and Use Committee at Cincinnati Children&#39;s Hospital Medical Center (CCHMC).
1. Preparation of Solutions
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	<li>Prepare egg water.
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		<li>Prepare the stock salt solut...

<ol>
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The current protocol describes a detailed procedure for acute ethanol treatment in zebrafish larvae and the subsequent histopathological analyses with H&#38;E staining. Acute ethanol treatment should be conducted at no earlier than 96 h post-fertilization, as this is the stage at which the zebrafish liver starts to express alcohol-metabolizing enzymes13. 2% ethanol is the maximal dose that larvae can tolerate13,14. The ethanol-treated larv...

Alcoholic Liver Disease (ALD), which is caused by alcohol overconsumption, is a major cause of alcohol-related morbidity and mortality. In the United States, nearly half of liver disease deaths involve alcohol1, and ALD is responsible for almost 1 in 3 liver transplants2. ALD has a broad spectrum. Steatosis, which is characterized by excess lipid accumulation in hepatocytes, occurs in the early stage of heavy drinking and is reversible upon cessation of alcohol use. Under the influence of genetic and environmental factors and continuing alcohol intake, hepatic steatosis can progress to alcoholic hepatitis and, eventually, cirrhosis 3. Studies using the rodent ALD models have provided substantial insights into the disease, but they have limitations (reviewed in reference3). Oral feeding of an alcohol diet only causes steatosis in rodents4,5. Development of inflammation and fibrosis requires either a second insult6,7 or chronic intragastric infusion, which is invasive and technically challenging8,9. The teleost zebrafish also develops liver injury in response to both chronic and acute alcohol treatment10,11,12,13,14,15. In particular, the larval zebrafish represents an attractive complementary model organism in which to study acute alcoholic liver injury10,11,13,15. The zebrafish liver is functional and produces key enzymes for ethanol metabolism by 4 days post-fertilization (dpf)13,16,17.Ethanol can be directly added to the water, and exposure to 2% ethanol for 24 h is sufficient to induce hepatic steatosis and fibrogenic responses in zebrafish larvae13,15.
It has been reported that treatment with 2% ethanol for 24 h resulted in a tissue ethanol concentration of 80 mM in zebrafish larvae13. Others have shown that larvae tolerate this concentration and the liver phenotypes seen in the treated animals are specific to ethanol exposure11,13,15,18. However, because 80 mM is nearly lethal in humans19, it is important to evaluate the liver histology of the ethanol-treated zebrafish and determine the physiological relevance to humans.
The rapid external development and translucence of zebrafish larvae make it possible to characterize the action of alcohol within the liver in real-time and in fixed samples. The availability of cell type-specific fluorescent transgenic lines and the recent advances in confocal microscopy facilitate the study of how different liver cell types change their morphology and behavior in response to acute ethanol treatment11,15. However, confocal imaging of the fluorescent transgenic zebrafish cannot completely substitute for Hematoxylin and Eosin (H&#38;E) staining when studying liver histology. Marking all liver cell types at the same time using transgenic zebrafish requires the generation of individual transgenic lines, each labeling one liver cell type with a unique fluorophore. Introducing different transgenic backgrounds into the same fish requires breeding multiple generations, which is time-consuming and costly. Additional immunofluorescence staining is needed to detect extracellular matrix components. H&#38;E staining, on the other hand, simultaneously labels all liver cell types and extracellular matrix components, thus providing an overview of the liver20. Moreover, it readily reveals several histopathological features of liver diseases, such as hepatocyte death, steatosis, and fibrosis. Although H&#38;E is a routine stain in mammalian liver histology, it is not commonly used in zebrafish liver research, and the protocol is less well established.
This work describes a protocol for acute ethanol treatment in zebrafish larvae and for the follow-up histological analyses with H&#38;E staining. The H&#38;E staining protocol can be used in all studies of liver development and function. Moreover, the paraffin sections can be used for immunohistochemistry, as well as for other special stains in liver pathology, including the trichrome stain, reticulin stain, etc.

<table border="0" cellpadding="0" cellspacing="0" width="1137">
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		<tr height="21">
			<td height="21" style="height:21px;width:645px;">1.5 mL centrifuge tubes</td>
			<td style="width:207px;">E &#38; K Scientific</td>
			<td style="width:119px;">280150</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>VWR International</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tubes</td>
			<td>VWR International</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% ethanol</td>
			<td>Decon Labs, Inc.</td>
			<td>2801</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid</td>
			<td>Newcomer Supply</td>
			<td>10010A</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Agarose</td>
			<td style="width:207px;">Research Products International</td>
			<td style="width:119px;">9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Aluminum jar rack holder</td>
			<td style="width:207px;">Newcomer Supply</td>
			<td style="width:119px;">5300JRK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacteriological petri dishes with lid</td>
			<td>Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Biopsy pads</td>
			<td style="width:207px;">Simport</td>
			<td style="width:119px;">M476.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Charged slides</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td>12-550-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Clear mounting media</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">8310-16</td>
			<td>Can be substituted with other clear mounting media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Commercial sea salts</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable microtome blades</td>
			<td>Fisher Scientific</td>
			<td>4280L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica Biosystems</td>
			<td>Leica Mz 95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Enclosed tissue processor</td>
			<td>Leica Biosystems</td>
			<td>ASP300 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin-Phloxine stain set</td>
			<td>Newcomer Supply</td>
			<td>1082A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Ethyl alcohol</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7023</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution, ACS reagent, 37 WT. % in H20, contains 10-15% methanol as stabilizer (to prevent polymerization)</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>A suspected carcinogen; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Formalin, Buffered, 10%</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">SF100-4</td>
			<td>A suspected carcinogen; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Graduated media bottle</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">16159-520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harris hematoxylin</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s212</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Histology molds</td>
			<td style="width:207px;">Sakura Finetek USA Inc</td>
			<td style="width:119px;">4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Hot plate/Stirrer</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">47751-148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Incubator</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">97058-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Insulin syringes</td>
			<td style="width:207px;">BD Medical</td>
			<td style="width:119px;">BD-309301</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:645px;">Inverted compound microscope</td>
			<td style="width:207px;">Carl Zeiss Microscopy</td>
			<td style="width:119px;">491912-9850-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Newcomer Supply</td>
			<td>12094E</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome</td>
			<td>Leica Biosystems</td>
			<td colspan="2">Leica Jung BioCut 2035&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nutating mixer</td>
			<td>VWR International</td>
			<td>82007-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Paraformaldehyde</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148-1KG</td>
			<td>A suspected carcinogen; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Pasteur pipet</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">53283-916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Pipette pump (10 mL)</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">53502-233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium phosphate, monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Grainger</td>
			<td>4A807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Slide Staining Kit</td>
			<td style="width:207px;">Newcomer Supply</td>
			<td style="width:119px;">5300KIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher BioReagents</td>
			<td>S318-500</td>
			<td>Very hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate, dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel strainer (5 inch diameter)</td>
			<td>Adaptive Science Tools</td>
			<td>L0906045in</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Tissue cassettes</td>
			<td style="width:207px;">Simport</td>
			<td style="width:119px;">M505.12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Tissue embedding center</td>
			<td style="width:207px;">Sakura Finetek USA Inc</td>
			<td style="width:119px;">#5100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue wipers, 1-Ply</td>
			<td>Fisher Scientific</td>
			<td>06666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Transfer pipets</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">137117M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine powder/Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base, primary standard and buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Wash bottle, low-density polyethylene, wide mouth</td>
			<td style="width:207px;">Nalge Nunc International</td>
			<td style="width:119px;">2402-0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Xylenes</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">X3S-4</td>
			<td>Irritant</td>
		</tr>
	</tbody>
</table>

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.

10% buffered formalin and 4% paraformaldehyde (PFA) are two of the most common fixatives used for histology practices. However, they do not give optimal fixation results for zebrafish liver tissue (Figure 1 and Table 1). Fixation with 10% formalin or 4% PFA often results in shrinkages, creating large gaps between the liver and surrounding tissues (Figure 1A,&#160;B; Figure 1B...

Watch this Scientific Journal Video about Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish at JoVE.com

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

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 ...

The overall goal of this experiment is to evaluate the patho-histology of the zebrafish larva liver caused by acute ethanol insults. This method can help address key questions in the field of alcoholic liver disease such as the histological feature of acute alcohol liver injury and to what extent the responses of zebrafish liver to acute alcohol injury resembles those seen in humans. The main advantage of this technique is that our ethanol treatment induces more robust responses than other published methods.Also, our histology protocol improves tissue integrity on the sections. Begin by selecting up to 40 96-hour post-fertilization larvae with inflated swim bladders using a Pasteur pipette. Split them evenly into two new Petri dishes.Remove as much residual egg water as possible, then add egg water containing 2%ethanol to one dish at a density of one larva per milliliter. Add the same amount of egg water without ethanol to the other dish to serve as the control. Keep the control and ethanol-treated larvae in the fish room for 24 hours.After the 24-hour period has elapsed, collect the larvae in a 1.5-milliliter centrifuge tube with no more than 20 larvae per tube. While working in the chemical hood, remove as much liquid as possible from the larvae, and then add at least one milliliter of Dietrich's fixative. Allow the fish to fix on a nutator at room temperature for at least 24 hours.Set out the tissue cassettes and two blue biopsy pads per cassette. Place one biopsy pad at the bottom of each cassette. Label each cassette in pencil clearly identifying the sample.While working in the fume hood, remove the fixative from the tubes, and wash the larvae three times for five minutes each time with one milliliter of PBS. Place the tubes on a nutator at room temperature while washing. During the washes, heat 3%agarose in water using a microwave to melt the solid.Heat for 30 seconds at a time, and watch carefully to minimize boiling. Once dissolved, place the liquid agarose on a hot plate set to 90 degrees Celsius with gentle stirring. Using a transfer pipette, transfer up to eight larvae to a plastic histology mold, and remove as much PBS as possible.Using a transfer pipette with the tip cut off, completely fill the mold with 3%agarose, then use an insulin syringe to gather the larvae to the center of the mold, and push them to the bottom. For sagittal sections, position the larvae in a line with the heads toward the top of the mold. To ensure that the livers are oriented in a consistent manner, turn the larvae so that the left side of the body is facing down, and flat against the bottom of the mold.After allowing the agarose to set for four to five minutes, remove the agarose block from the mold, and use a razor blade to trim the agarose around the larvae. Stand the block on end, and cut the thickness in half so that the final block is roughly two to three millimeters thick. Transfer the small agarose block to the prepared tissue cassette, and place the second biopsy pad on top of the block.Close the cassette, and place the fully assembled cassette into a sealable container with freshly prepared 70%ethanol in double-distilled water. After paraffin embedding the tissue, use a microtome to remove excess paraffin covering the tissue by sectioning five microns at a time until the tissue is exposed at the surface of the paraffin block, then stop and discard all the sections that are cut. Soak the blocks face down in PBS, and place it four degrees Celsius overnight.Then next day, remove one block at a time from the PBS, and cut five micron sections using a microtome. Separate the ribbon of sections from the blade by gently pulling the last section away from the blade using forceps or a brush. Use the forceps to pick up the ribbon by the last section, and transfer it to a water bath at 42 degrees Celsius.Allow the ribbons to float on the surface of the water for at least five minutes. Use the forceps to tease apart sections into smaller groups to fit onto the slides. Put a charged slide into the water at a 45-degree angle, and carefully position it underneath the group of sections to be collected.Carefully lift the slide from the water, and allow the sections to attach to the slide. Blot any excess water from the sections using lint-free tissue. Place the slide in a slide holder or box.Continue to section the block until the desired tissue has been collected. When finished, bake the slides in a 55 degrees Celsius incubator or oven for three to 16 hours to melt the paraffin. After the allotted time, check that the paraffin has melted, and allow the slides to cool before staining.Deparaffinize the slides by immersing them in 100%xylene twice for 15 minutes each time, then rehydrate the slides through an ethanol gradient. For each solution, dip eight to 10 times for two seconds per dip until the liquid runs cleanly off the slides. Place the slides in 100%filtered Harris Hematoxylin for four minutes.After four minutes, quickly transfer them back to the container of deionized water. Run deionized water into the back corner of the container farthest away from the sections. Empty the container periodically until the water is no longer purple.Quickly check the Hematoxylin intensity on a dissecting microscope using gooseneck lights. Return the slides to 100%Hematoxylin for one minute if further staining is required. Once the desired Hematoxylin staining has been obtained, dip the slides twice in 0.05%hydrochloric acid, then immediately transfer them back to the container with clean deionized water.Empty and refill the container with water twice. Transfer the slides to two containers of 95%ethanol for 30 seconds each. Place the slides into the Eosin Y-Phloxine B solution for two minutes.After the two minutes, transfer the slides back to the previous 95%ethanol container, then check the intensity of the color under the dissecting microscope. If the staining is not sufficient, return to the Eosin solution for 30 seconds. Repeat as necessary.When staining of the desired intensity has been obtained, transfer the slides to 100%isopropanol for 15 seconds. Replace with fresh 100%isopropanol, and place the slides back in the isopropanol for another 15 seconds. Repeat this process for a total of six isopropanol washes.After immersing in 100%xylene for three minutes, remove one slide. Add sufficient mounting medium to cover the sections, and then dip the slide in 100%xylene. Apply a cover slip to the slide, and blot any excess mounting medium on a paper towel until only a thin line is seen, then dip a tissue into the xylene, and wipe the back of the slide to remove any medium that has dripped.Place the slide flat on a sturdy but mobile surface like a piece of cardboard, and allow the xylene to evaporate in the hood for 10 minutes. Leave the slides at room temperature overnight to allow the mounting medium to harden before imaging. This 120 hours post-fertilization zebrafish larvae liver was immersed in Dietrich's fixative at room temperature for 24 hours.Dietrich's fixative provides the optimal fixation of this tissue. With 10%formalin fixation, the hepatocytes often lose their cytoplasm. The tissue is not stained properly with Eosin, and appears purple.After fixation with 4%PFA, gaps are seen between the hepatocytes. 4%PFA causes the liver tissue to shrink so there appears to be a large gap between the liver and the surrounding tissues. The dashed line marks the border of the surrounding tissues.This higher power image shows H&E staining of the liver in a wild-type untreated control larva that was part of an ethanol treatment study. Again, this animal was fixed with Dietrich's fixative at 120 hours post-fertilization. This image shows the effect of treatment with 2%ethanol from 96 to 120 hours post-fertilization on the liver.This acute ethanol treatment causes hepatic steatosis, and blood vessel swelling. The arrows point to the hepatocytes with excessive deposition of round droplets, and the asterisks mark the swollen hepatic blood vessels. While attempting this procedure, it's important to remember to perform the ethanol treatment in a fish facility with a light-dark cycle.For H&E, use Dietrich's fixative for an optimal tissue preservation. Also remember to check the color intensity frequently to avoid over-staining or under-staining.

histological-analyses-of-acute-alcoholic-liver-injury-in-zebrafish

Research

JoVE Journal

Developmental Biology

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.

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 ...

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 ...

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

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 ...

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 ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...