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 c...

<ol>
	<li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+liver+regeneration+and+growth+homeostasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+it+new+again:+insight+into+liver+development,+regeneration,+and+disease+from+zebrafish+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration:+biological+and+pathological+mechanisms+and+implications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+growth+in+the+embryo+and+during+liver+regeneration+in+zebrafish+requires+the+cell+cycle+regulator+uhrf1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APC+mutant+zebrafish+uncover+a+changing+temporal+requirement+for+wnt+signaling+in+liver+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+II+is+required+for+embryonic+development+and+liver+regeneration+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+growth+mechanisms+regulated+by+BMP+and+FGF+signaling+mediate+liver+regeneration+in+zebrafish+after+partial+hepatectomy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haploinsufficiency+of+Def+activates+p53-dependent+TGF&#946;+signalling+and+causes+scar+formation+after+partial+hepatectomy.">Haploinsufficiency of Def activates p53-dependent TGF&#946; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+characterization+of+the+dorsal+lobes+after+hepatectomy+of+the+ventral+lobe+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizational+principles+of+the+liver.">Organizational principles of the liver.</a> The Liver: Biology and Pathobiology: Fifth Edition. , 1-15 (2009).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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&#34; 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

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...

Watch this Scientific Journal Video about Partial Hepatectomy in Adult Zebrafish at JoVE.com

Partial Hepatectomy in Adult Zebrafish

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 ...

This protocol will allow researchers to take advantage of available chemical and genetic tools to study the mechanisms of liver regeneration in zebrafish. Partial hepatectomy allows the precise removal of tissue to stimulate liver regeneration without the need for the body-wide effects of toxic liver injury. Only by understanding the molecular and cellular mechanisms of liver regeneration can we design new therapies to treat patients with advanced liver disease.To begin, remove the fish from the Tricaine tank using forceps and embed them in the groove of a Tricaine soaked sponge with the ventral side up. Then place the sponge under a dissecting microscope with top-down illumination. Pinch the skin and scales just posterior to the heart with fine forceps and create a hole in the body cavity by making a cut under the forceps with the spring loaded scissor.Rotate the sponge and make a three to four millimeter incision along the abdomen by cutting posterially toward the pelvic fins. Then rotate the sponge back. 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 is present on top of the intestine, and the liver is a pink or orange structure spread out over the golden brown intestine, and can be clearly visualized with CFP fluorescence. Squeeze the fine forceps so that the tines are touching each other and slide the tines between the liver and the intestine. Then slowly separate the tines to break the portal vein attachments between the liver and the intestine.Repeat this process until all the portal connections between the liver and intestine have been severed, and pull the ventral lobe from the intestine using fine forceps. Cut the ventral lobe free from the rest of the liver. Examination of the animal with CFP fluorescence demonstrates that the ventral lobe has been removed.When finished, carefully transfer the fish from the sponge into the tank containing system water and stream the system water over the gills for a few minutes until the fish recovers and swims on its own. After euthanizing the fish, remove it from ice water. Place it in the groove of a sponge with the ventral side up and make an incision of the ventral body wall at the anterior posterior position of the heart.Then make two more incisions from the edges of the first incision that run along the anterior posterior axis to the pelvic fins, and peel back the skin and muscle to reveal the visceral organs. The visceral organs can now be visualized with both bright field and fluorescence microscopy. Liver recovery after partial hepatectomy was estimated by calculating the liver to body weight ratio, which was 1.8%for males and 3.3%for females.The liver to body weight ratio of both sexes was determined at post-surgery day zero, and day seven in partial hepatectomy and sham controls. At post-surgery day zero, sham animals have a visible ventral lobe, but in partial hepatectomy animals, the ventral lobe was completely absent, which resulted in a 30%reduction in males and 20%reduction in females of liver to body weight ratios. At post-surgery day seven in partial hepatectomy animals, the ventral lobe was not regenerated, and the liver to body weight ratios in partial hepatectomy and sham controls were comparable, indicating regained liver mass after partial hepatectomy.The effect of time on ventral lobe regeneration was determined by performing a full 1/3 partial hepatectomy and measuring the ventral lobe to the intestine length ratio. The ventral lobe occupied 50 to 100%of the intestine length in sham controls, but was severely reduced in 1/3 partial hepatectomy animals. Partial hepatectomy animals at post-surgery day 36 had an increased ventral lobe to intestine ratio as compared to post-surgery day one.A statistically significant increased size of the ventral lobe was observed in animals recovering from partial hepatectomy at post-surgery day 36 as compared to day one, but not in sham controls. Some animals regenerated the ventral lobe, but some did not, indicating variation in surgery response. The most critical part of this protocol is to cleanly separating the liver and the intestine so that the ventral lobe can be entirely removed.Partial hepatectomy in zebrafish can be performed in mutant or transgenic lines to assay that genetic requirements for liver regeneration.

Research

JoVE Journal

Biology

3.7K visualizações.  Harvard Medical School. Este protocolo permitirá que os pesquisadores aproveitem as ferramentas químicas e genéticas disponíveis para estudar os mecanismos de regeneração hepática em zebrafish. A hepatectomia parcial permite a remoção precisa do tecido para estimular a regeneração hepática sem a necessidade dos efeitos em todo o corpo da lesão hepática tóxica. Somente entendendo os mecanismos moleculares e celulares da regeneração hepática podemos projetar novas terapias para tratar pacientes com d...