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/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 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 vein that then splits as it enters the liver12. In contrast, the zebrafish liver receives portal circulation from a series of tiny vessels that move directly from the intestine into the liver (Figure 2B). Thus, each lobe of the liver, flattened out over the intestine, is securely attached to the intestine by these vessels. Attempting to remove the ventral lobe without first severing the portal veins can often result in incomplete resection (data not shown). Some of the initial protocols describe pulling the ventral lobe out through a relatively minor incision, which does not allow for severing these portal attachments5,6.
The protocol described here is designed to address these challenges to enable consistent ventral lobe removal. Anesthetized zebrafish are placed snugly in the groove of sponge to keep their gills wet and their bodies immobile for the duration of the procedure. Pinching the skin just posterior to the heart makes it possible to place an incision in the skin without damaging any of the internal organs. Opening a large (3-4 mm) incision makes removal of the liver and assessing the degree of removal straightforward (Figure 2C). Importantly, zebrafish can recover from and ultimately heal a wound of this size without any primary wound closure. By squeezing the sponge and compressing the body wall, the visceral organs become more accessible and it is possible to slide the tines of a forceps in between the liver and the intestine. The forceps can then be used to sever the portal connections between the liver and the intestine. Once this has been achieved, fine forceps can be used to peel back the ventral lobe so that it can be separated from the rest of the liver.
We measured the liver to body weight ratios (LBR) of animals subjected to sham and PHX surgeries. We found that by 7 dpi, the LBR of PHX animals was comparable to controls (Figure 3F). Given that the ventral lobe had not regenerated at this point (Figure 3E), we inferred that the regeneration occurred through compensation in the dorsal lobes. To address the question of whether the ventral lobe can ultimately regenerate in zebrafish, we performed PHX and examined liver re-growth. On average, the ratio of the length of the ventral lobe to the intestine was higher at 36 dpi than at 1 dpi, indicating regeneration of the ventral lobe (Figure 4C-D). There is a wide variation in animal response to the injuries, with some animals experiencing very little growth and others experiencing substantial recovery of the ventral lobe (Figure 4E). We conclude that the liver can regenerate with a mixture of epimorphic regeneration of the ventral lobe and compensatory regeneration in the dorsal lobes, albeit at different timescales. As prior studies have used partial hepatectomies to assay to role of individual genes5,7,9 and signaling pathways6,8,10 after resection, we anticipate that this protocol will advance our understanding of the molecular and cellular mechanisms of liver regeneration in an adult vertebrate.

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

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 tissue. The survival rates for sham controls ranged from 90%-100% for both male and female zebrafish. The survival rates for PHX animals ranged from 60%-75% for male zebrafish and 60%-90% for female zebrafish, with all deaths occurring in the first 24 h following the surgery.
The gold standard for quantifying the recovery of the liver following PHX is the liver to body weight ratio, or LBR. This assay has been used for both measurement of liver mass recovery in both mammalian11 and zebrafish models8. To reliably quantify the weight of the liver, we made weight measurements on fixed animals, as previously described8. We performed LBR measurements on wild type, uninjured zebrafish, and found that as previously reported8, female zebrafish had almost double the LBR as compared to male zebrafish (3.3% for female zebrafish and 1.8% for male zebrafish) (Figure 3A). We performed both sham and PHX on adult zebrafish of both sexes and analyzed the LBR at 0 and 7 days post injury (dpi) (Figure 3B). At 0 dpi, sham animals had a clearly visible ventral lobe, whereas in PHX animals the ventral lobe was completely absent (Figure 3C). Notably, this resulted in a significant reduction in LBR (30% reduction in male fish, 20% reduction in female fish) (Figure 3D). In the cohort of fish analyzed at 7 dpi, we found that the ventral lobe had not regenerated (Figure 3E), and yet the LBR of PHX and sham controls were comparable (Figure 3F). These measurements indicate that by 7 dpi, the liver has regained mass relative to sham controls, presumably by compensatory regeneration in the dorsal lobes, in agreement with prior reports8,9.
We decided to investigate whether, given enough time, the ventral lobe of the liver was capable of regeneration. Adult Tg(fabp10a:CFP-NTR) fish were selected for experimentation, as these fish express CFP in hepatocytes, allowing for visualization of the liver using fluorescence microscopy. A cohort of animals was subjected to the full one-third partial hepatectomy protocol. Animals were analyzed at 1 or 36 dpi (Figure 4A). For each animal, the ratio of ventral lobe length to the intestine was measured (Figure 4B).
Animals subjected to sham surgeries had a prominent ventral lobe that occupied 50%-100% of the intestine length, whereas animals subjected to one-third partial hepatectomy had a severely reduced ventral lobe. Strikingly, many partial hepatectomy animals at 36 dpi had an increased ventral lobe to intestine ratio as compared to partial hepatectomy animals at 1 dpi (Figure 4C). There was no increase in the size of the ventral lobe in sham controls; however, a statistically significant increase in the size of the ventral lobe was observed after recovery from partial hepatectomy (Figure 4D). We noted that there was a wide variation in the response to the surgery, with some animals displaying no regeneration, and others clearly regenerating a well-defined ventral lobe (Figure 4E). These results demonstrate that the ventral lobe is capable of regenerating from surgical liver resection in adult zebrafish. Taken together, our work indicates that the zebrafish liver is capable of both epimorphic and compensatory regeneration.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62349/62349fig01.jpg" /> 
Figure 1: The partial hepatectomy protocol. (A) Diagram of an adult zebrafish, with the anterior-posterior and dorsal-ventral axes noted. (B) After anesthetization, an animal is embedded in a sponge (shown in blue) so that it is ventral side up. Fine forceps are used to pinch the skin just posterior to the heart. The sponge is not shown in the subsequent panels for clarity. (C) Scissors are used to break the skin just posterior to the heart. (D-E) The scissors are used at the now open wound to make a 3-4 mm incision running from anterior to posterior, terminating just before the pelvic fins. (F) Squeezing the sides of the sponge causes the visceral organs to pop out of the larger wound. (G) Fine forceps with both tines touching are slid between the ventral lobe of the liver and the intestine. (H) The forceps are relaxed, causing them to break the portal vein attachments between the liver and the intestine. This process is repeated along the length of the ventral lobe. (I-J) The ventral lobe is pulled up from its posterior end, while the scissors are used to sever the ventral lobe from the rest of the liver. (K) The animal is placed into a recovery chamber with system water, where it regains consciousness and rights itself. (L) With time, the viscera are pulled back into the body cavity and the wound heals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62349/62349fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62349/62349fig02.jpg" /> 
Figure 2: Zebrafish anatomy and recovery from partial hepatectomy. (A) For analysis of the visceral organs, animals are euthanized and placed ventral side up on a sponge. First, an incision is performed at the anterior-posterior position of the heart (red line). Then, two incisions are generated that run along the anterior-posterior axis to the pelvic fins (blue line). Then, the skin and muscles are peeled back, revealing the visceral organs. Labeled are the intestine, ventral lobe of the liver, and the central vein in that lobe. (B) Live image of a ventral view of a zebrafish prepped for analysis of the visceral organs. The ventral lobe of the liver is outlined with a dotted white line. Yellow box in the 2x image indicates the location of the 8x image. White arrowheads indicate portal vein connections between the intestine and the liver. (C) Ventral view of the wound generated by the partial hepatectomy procedure at the indicated timepoints. Over time, the incision heals completely. Scale bars, 500 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62349/62349fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62349/62349fig03.jpg" /> 
Figure 3: Compensatory regeneration after partial hepatectomy. (A) Liver to body weight (LBR) ratios were measured for wild type, uninjured zebrafish. Female zebrafish have almost double the LBR compared to male zebrafish. Female animals were compared to male animals using a Wilcoxon rank sum test, ****p &#60; 0.0001. (B) Schematic indicating that wild type zebrafish were used for this experiment. Animals were subjected to sham or partial hepatectomy (PHX), and then either fixed at 0 or 7 days post injury (dpi). Fixed animals were imaged and subjected to LBR measurements. (C,E) Shown are representative images of animals subjected to sham or PHX at 0 dpi (C) and 7 dpi (E). Note the complete absence of the ventral lobes in PHX animals. For all images, shown is a ventral view of the visceral organs. Images are bright-field images. The ventral lobe of the liver is outlined in white. Scale bars, 500 &#956;m. (D,F) Bar graphs of the liver to body weight ratios for both male and female zebrafish after sham and PHX surgeries at 0 dpi (D) and 7 dpi (F). The height of the bar is the mean value, and the error bars represent SEM. Whereas there is a reduction in LBR following partial hepatectomy at 0 dpi, there is no significant difference between PHX and sham animals at 7 dpi, indicating restoration of LBR. Partial hepatectomy samples were compared to sham controls using a Wilcoxon rank sum test, ns = not significant, *p &#60; 0.05, **p &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62349/62349fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62349/62349fig04.jpg" /> 
Figure 4: Zebrafish can regenerate their ventral lobes after partial hepatectomy. (A) Schematic indicating that Tg(fabp10a:CFP-NTR) were used for this experiment. Surgery and analysis by imaging were performed at the indicated timepoints. (B) An example image to demonstrate how animals were analyzed. Animals were quantified by measuring the length of the ventral lobe (in red), the length of the intestine (in yellow), and computing the percentage of the intestine that the ventral lobe occupies (in white). (C) Shown is a representative image of animals subjected to sham or partial hepatectomy (PHX) at 1 and 36 days post injury (dpi). (D) Violin plots of the ratio of ventral lobe length to intestine length for animals subjected to sham or partial hepatectomy at 1 and 36 dpi. Each dot represents the value from a single fish. Values for male fish are in blue, female fish in red. Partial hepatectomy samples were compared to sham controls using a Wilcoxon rank sum test, ns = not significant, *p &#60; 0.05. (E) Shown are two examples of partial hepatectomy animals at 36 dpi that did not regenerate, and two examples that did regenerate. For all images, shown is a ventral view of the visceral organs. Images are a merge of a CFP fluorescence image and a bright-field image. CFP fluorescence is only present in the liver. The ventral lobe of the liver is outlined in red. The percentage of the intestine that the ventral lobe occupies is in white. Scale bars, 500 &#956;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62349/62349fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

Nanyang Technological University

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Biology

3.9K Views.  Harvard Medical School. Dit protocol stelt onderzoekers in staat om gebruik te maken van beschikbare chemische en genetische hulpmiddelen om de mechanismen van leverregeneratie bij zebravissen te bestuderen. Gedeeltelijke hepatectomie maakt de nauwkeurige verwijdering van weefsel mogelijk om leverregeneratie te stimuleren zonder de lichaamsbrede effecten van toxisch leverletsel. Alleen door de moleculaire en cellulaire mechanismen van leverregeneratie te begrijpen, kunnen we nieuwe therapieën ontwerpen om patiënte...

Video: Partial Hepatectomy in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

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

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

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 ...

Developmental Biology

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 ...

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 ...

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

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

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.

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Cancer Research

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 ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

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.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Dissection of the Adult Zebrafish Kidney

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

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

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

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

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.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

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

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

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.

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

Partial Hepatectomy in Adult Zebrafish

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