Name: Partial Hepatectomy in Adult Zebrafish (Video) | JoVE Journal
Uploaded: 2021-04-04T15:00:00.000+00:00
Duration: 6 min 6 s
Description: Learn partial hepatectomy in adult zebrafish to study liver regeneration mechanisms. This reproducible protocol enables precise tissue removal and molecular investigation of liver regrowth using Danio rerio.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

I.M.O. is supported by the NIAAA (F32AA027135). W.G. is supported by R01DK090311, R01DK105198, R24OD017870, and the Claudia Adams Barr Program for Excellence in Cancer Research. W.G. is a Pew Scholar in Biomedical Sciences.

Zebrafish were raised and bred according to standard procedures. Experiments were approved by the Brigham and Women&#39;s Hospital&#39;s Institutional Animal Care and Use Committee (2016N000405). Adult zebrafish were fasted for 24 h prior to the start of the protocol. System water refers to the water in zebrafish housing tanks in the aquatic facility.
1. Preparation and anesthetization
<ol>
	<li>Prepare 0.016% Tricaine solution in system water. 
	CAUTION: Tricaine is an irritant if it comes in contact with the eyes, skin, or respiratory tract.</li>
	<li>Prepare a sponge to hold anesthetized zebrafish during the dissection protocol. Cut a full sponge into quarters. Using a razor blade, remove a thin wedge of sponge that runs parallel to the long axis of the sponge quarter.
	<ol>
		<li>The slit should be long enough to accommodate an adult fish (this will vary between different sizes of fish). For example, for an adult fish 35 mm in length, the length of the slit should be 20 mm. The head and body are snugly held in the sponge, but the tail runs past the edge of the sponge (Figure 1B).</li>
	</ol>
	</li>
	<li>Soak the sponge in 0.016% Tricaine solution.</li>
	<li>Place adult zebrafish (either male or female) in 625 mL of 0.016% Tricaine solution.</li>
	<li>Incubate for 6 min or until the fish is unresponsive to touch.</li>
	<li>Using forceps, carefully remove the fish from the Tricaine tank and place the fish ventral side up in the groove of the sponge (Figure 1A - B).</li>
	<li>Place the sponge under a dissecting microscope with top-down illumination.</li>
</ol>
2. Surgery
<ol>
	<li>Using fine forceps, pinch the skin and scales medially, just posterior to the heart (Figure 1B).</li>
	<li>Using spring-loaded scissors, make a cut under the forceps to create a hole in the body cavity (Figure 1C - D). Take care not to injure the heart or a major blood vessel, as this will result in increased mortality.</li>
	<li>Using spring-loaded scissors, make a 3-4 mm incision along the abdomen, processing posteriorly until the incision arrives at the pelvic fins (Figure 1D - E). By this point, the ventral lobe of the liver may be visible through the incision.</li>
	<li>Squeeze the sides of the sponge with one hand to force the visceral organs out of the body cavity. The ventral lobe of the liver will be visible on top of the intestine (Figures 1F,2A-B). The liver will appear as a pink or orange structure spread out over the golden-brown intestine. Animals designated as sham controls are recovered at this point.</li>
	<li>Squeeze the fine forceps so that the two tines are touching. While keeping pressure on the sponge, slide the tines of the fine forceps in between the liver and the intestine (Figure 1G). Take care to not puncture the intestine, as this will result in increased mortality.</li>
	<li>Slowly relax the pressure on the forceps so that the tines move away from one another (Figure 1H). This sliding action severs the numerous portal vein attachments between the ventral lobe and the intestine (Figure 2B), and is necessary to cleanly remove the ventral lobe. Repeat this process until all of the portal connections between the liver and intestine have been severed.</li>
	<li>Peel back the ventral lobe from the intestine using fine forceps and cut the ventral lobe free from the rest of the liver (Figure 1I).</li>
	<li>This procedure results in a one-third partial hepatectomy (Figure 1J).</li>
</ol>
3. Recovery
<ol>
	<li>Carefully remove the fish from the sponge and place it in a tank of system water.</li>
	<li>Pipette system water over the gills for a few minutes until the fish is swimming on its own (Figure 1K).</li>
	<li>Monitor fish for 2-4 hours before placing them back onto the system. Do not feed the fish for a full 24 h after the surgery.</li>
	<li>Monitor fish daily for the duration of the experiment.</li>
	<li>With time, the incision in the body wall will heal naturally without the need for sutures (Figure&#160;1L,2C).</li>
</ol>
4. Ventral lobe to intestine length analysis
<ol>
	<li>Euthanize all animals destined for analysis in ice water for 10 min until all opercular movements cease.</li>
	<li>Remove the fish from ice water and place it ventral side up in the groove of a sponge.</li>
	<li>Using spring-loaded scissors, make an incision in the ventral body wall at the anterior-posterior position of the heart. Then make two more incisions that run along the anterior-posterior axis from the first incision all the way to the pelvic fins. (Figure 2A).</li>
	<li>Peel back the skin and muscle to reveal the visceral organs (Figure 2A).</li>
	<li>Acquire bright-field and fluorescent images of the visceral organs using an epifluorescence microscope. This field of view will include the area where the ventral lobe was resected. Because animals are euthanized prior to analysis, this kind of analysis precludes long-term imaging of the same fish.</li>
</ol>
5. Liver to body weight ratio analysis
<ol>
	<li>Euthanize all animals destined for analysis in ice water for 10 min until all opercular movements cease.</li>
	<li>Place fish in a 50 mL conical tube.</li>
	<li>Add 25 mL of 4% paraformaldehyde in 1x PBS and 0.3% Tween to the tube. 
	CAUTION: Formaldehyde is toxic, and solutions containing formaldehyde should always be processed in a chemical hood.</li>
	<li>Nutate for 48 h at 4 &#176;C.</li>
	<li>Perform four 10 min washes in 1x PBS and 0.3% Tween.</li>
	<li>Retrieve fish with forceps, and blot dry on a paper towel.</li>
	<li>Record the weight of the entire fish.</li>
	<li>Using spring-loaded scissors, make an incision in the ventral body wall at the anterior-posterior position of the heart. Then, make two more incisions that run along the anterior-posterior axis from the first incision all the way to the pelvic fins. (Figure 2A).</li>
	<li>Peel back the skin and muscle to reveal the visceral organs (Figure 2A).</li>
	<li>Acquire bright-field images of the liver using an epifluorescence microscope.</li>
	<li>Dissect out the liver, placing the pieces of liver on a weighing boat.</li>
	<li>Record the weight of the liver.</li>
</ol>

<ol><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+liver+regeneration+and+growth+homeostasis%5BTitle%5D)+AND+%22Comprehensive+Physiology%22%5BJournal%5D)">Principles of liver regeneration and growth homeostasis.</a> Comprehensive Physiology. 3, 485-513 (2013).</li>
<li>Wang, S., Miller, S. R., Ober, E. A., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+it+new+again%3A+insight+into+liver+development%2C+regeneration%2C+and+disease+from+zebrafish+research%5BTitle%5D)+AND+%22Current+Topics+in+Developmental+Biology%22%5BJournal%5D)">Making it new again: insight into liver development, regeneration, and disease from zebrafish research.</a> Current Topics in Developmental Biology. 124, (2017).</li>
<li>Michalopoulos, G. K., Bhushan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+regeneration%3A+biological+and+pathological+mechanisms+and+implications%5BTitle%5D)+AND+%22Nature+Reviews+Gastroenterology+and+Hepatology%22%5BJournal%5D)">Liver regeneration: biological and pathological mechanisms and implications.</a> Nature Reviews Gastroenterology and Hepatology. , (2020).</li>
<li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+as+a+model+for+complex+tissue+regeneration%5BTitle%5D)+AND+%22Trends+in+Genetics%22%5BJournal%5D)">The zebrafish as a model for complex tissue regeneration.</a> Trends in Genetics. 29, 611-620 (2013).</li>
<li>Sadler, K. C., Krahn, K. N., Gaur, N. A., Ukomadu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+growth+in+the+embryo+and+during+liver+regeneration+in+zebrafish+requires+the+cell+cycle+regulator+uhrf1%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator uhrf1.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 1570-1575 (2007).</li>
<li>Goessling, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((APC+mutant+zebrafish+uncover+a+changing+temporal+requirement+for+wnt+signaling+in+liver+development%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development.</a> Developmental Biology. 320, 161-174 (2008).</li>
<li>Dovey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topoisomerase+II+is+required+for+embryonic+development+and+liver+regeneration+in+zebrafish%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Topoisomerase II is required for embryonic development and liver regeneration in zebrafish.</a> Molecular and Cellular Biology. 29, 3746-3753 (2009).</li>
<li>Kan, N. G., Junghans, D., Belmonte, J. C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compensatory+growth+mechanisms+regulated+by+BMP+and+FGF+signaling+mediate+liver+regeneration+in+zebrafish+after+partial+hepatectomy%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy.</a> The FASEB Journal. 23, 3516-3525 (2009).</li>
<li>Zhu, Z., Chen, J., Xiong, J. W., Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Haploinsufficiency+of+Def+activates+p53-dependent+TGF%CE%B2+signalling+and+causes+scar+formation+after+partial+hepatectomy%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy.</a> PLoS One. 9, (2014).</li>
<li>Feng, G., Long, Y., Peng, J., Li, Q., Cui, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptomic+characterization+of+the+dorsal+lobes+after+hepatectomy+of+the+ventral+lobe+in+zebrafish%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Transcriptomic characterization of the dorsal lobes after hepatectomy of the ventral lobe in zebrafish.</a> BMC Genomics. 16, 979(2015).</li>
<li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+regeneration%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">Liver regeneration.</a> Journal of Cellular Physiology. 213, 286-300 (2007).</li>
<li>Grisham, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organizational+principles+of+the+liver%5BTitle%5D)+AND+%22The+Liver%3A+Biology+and+Pathobiology%3A+Fifth+Edition%22%5BJournal%5D)">Organizational principles of the liver.</a> The Liver: Biology and Pathobiology: Fifth Edition. , 1-15 (2009).</li>
</ol>

The authors declare that they have no competing financial interests.

The anatomical differences between zebrafish and mammalian models for liver regeneration present unique challenges to liver resection. The liver in zebrafish is in close proximity to the heart and the intestine; inadvertently damaging either organ results in increased mortality. The zebrafish liver is not encapsulated, making it more difficult to separate from the intestine. The liver receives nutrient-rich blood from the intestine through portal veins. In mammals, veins leaving the intestine converge on a primary portal...

Among the solid organs in humans, the liver is the only organ capable of regeneration1. This is critical, as the liver is an essential organ, responsible for key metabolic functions, energy storage, blood detoxification, secretion of plasma proteins, and bile production2. Hepatocytes lost due to toxic or inflammatory damage are replaced primarily via division of the remaining hepatocytes1. One classical experimental model for studying liver regeneration is partial hepatectomy, where individual lobes of the liver are removed, leaving the remaining lobes intact3. This procedure was initially developed in rats, in which approximately two-thirds of the liver mass is removed. After partial hepatectomy in mammals, compensatory regeneration occurs in the remaining lobes until the liver recovers its initial mass. Notably, the mammalian liver does not replace the missing lobes.
Zebrafish (Danio rerio) represent a tractable model for studying adult organ regeneration4. The zebrafish liver, while structurally different from the mammalian liver, is made up of the same cell types and serves the same function as in mammals2. It is composed of three lobes, with two dorsal lobes and a single ventral lobe that are flattened along the intestine. Partial hepatectomy has previously been performed in zebrafish, with conflicting accounts as to the precise mode of regeneration. Typically, a one-third partial hepatectomy is performed by removal of the entire ventral lobe. Initial reports indicated that after removal of the ventral lobe, it was fully regenerated within a week5,6,7, suggesting that in contrast to the mammalian liver, the zebrafish liver is capable of epimorphic regeneration. Subsequent studies demonstrated that removal of the ventral lobe resulted in compensatory regeneration in the dorsal lobes, rather than the regeneration of the missing ventral lobe, and ultimately the recovery of liver mass within a week8,9. Transcriptomic profiling of the dorsal lobes following resection of the ventral lobe revealed significant changes associated with compensatory regeneration10. Given that the mode of liver regeneration can vary with the extent of the injury8, we speculated that the discrepancies in results may be due to technical variation in the partial hepatectomy protocol between research groups.
This protocol describes a procedure for performing a one-third partial hepatectomy on adult zebrafish by removing the ventral lobe. This technique will be valuable for assessing mechanisms of liver regeneration.

<table><tbody><tr><td>16% Paraformaldehyde Aqueous Solution, EM Grade</td><td>Electron Microscopy Sciences</td><td>15700</td><td></td></tr><tr><td>50 mL Falcon Centrifuge Tubes, Polypropylene, Sterile</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>AS 82/220.R2 PLUS Analytical Balance</td><td>Bay State Scale &amp;amp; Systems, INC.</td><td>WL-104-1051</td><td></td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>EMS Kuehne Coverglass/Specimen Forceps</td><td>Electron Microscopy Sciences</td><td>72997-07</td><td></td></tr><tr><td>Epifluorescence microscope</td><td>Zeiss</td><td>Discovery.V8</td><td></td></tr><tr><td>Mastertop Cellulose Cleaning Scrub Sponge</td><td>Amazon</td><td>B07CBSM53Z</td><td></td></tr><tr><td>PBS10X Liquid Conc 4L</td><td>EMD Millipore</td><td>6505-4L</td><td></td></tr><tr><td>Super Fine Micro Scissors, 3 1/4&quot; straight</td><td>Biomedical Research Instruments</td><td>11-1020</td><td></td></tr><tr><td>Tricaine methanesulfonate</td><td>Syndel</td><td>TRIC-M-GR-0010</td><td></td></tr><tr><td>Tween 20, Fisher BioReagents</td><td>Fischer Scientific</td><td>BP337-500</td><td></td></tr></tbody></table>

partial hepatectomy in adult zebrafish

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

In order to examine the regenerative potential of the adult zebrafish liver, we performed partial hepatectomy (PHX) in adult zebrafish. In general, large adults (30-40 mm in length) were selected, ranging from 1.5-2.5 years old. Within individual experiments, animals were selected from the same tank, and were age- and size-matched. As an appropriate control, we utilized sham surgeries in which the animal was both anesthetized and received a large incision in the ventral body wall but was recovered without removing any ti...

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

Research

JoVE Journal

Biology

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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

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.

Dissection of the Adult Zebrafish Kidney

The article describes targeted liver ablation, a method for inducing hepatocyte death with metronidazole in transgenic zebrafish larvae. The sample protocol describes the process for generating, manipulating and assessing targeted liver ablation in zebrafish.

Targeted Liver Ablation: Inducing Hepatocyte Death with Metronidazole in Transgenic Zebrafish Larvae

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recovery of Adult Zebrafish Hearts for High-throughput Applications

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.

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

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.

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

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.

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

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.

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

Learn partial hepatectomy in adult zebrafish to study liver regeneration mechanisms. This reproducible protocol enables precise tissue removal and molecular investigation of liver regrowth using Danio rerio.

Partial Hepatectomy in Adult Zebrafish

Explore More Videos