We thank P. Chu for performing H&#38;E and histology; and C. Wang and A. McCarty for helpful discussions. Research was supported through funding from the Virginia and D. K. Ludwig Fund for Cancer Research; the National Heart, Lung, and Blood Institute (R01HL058770 and U01HL099999); and the California Institute for Regenerative Medicine (RC1 00354) grants to I.W. Y.R. was supported by the Human Frontier Science Program Career Development Award (CDA00017), the German Research Foundation (RI 2787/1), the Siebel Stem Cell Institute, and the Thomas and Stacey Siebel Foundation (1119368-104-GHBJI). J.M.T. was supported by the NIH (T32GM007365), the National Research Service Award (1F30DK108561), and the Paul and Daisy Soros Fellowship for New Americans.

All animal experiments were carried out in strict accordance with the guidelines set forth by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and Stanford University&#39;s Administrative Panel on Laboratory Animal Care (APLAC), (Protocol number #10266) and in the United States, or the European Animal Welfare Act, Directive 2010/63/EU. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Government of Bavaria, Germany, and received the perm...

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Acute hepatic injury has traditionally been studied using chemical (acetaminophen, ethanol, carbon tetrachloride), or surgical models (70% partial hepatectomy). The regenerative response after 70% partial hepatectomy has been characterized to involve global hepatocyte hypertrophy and multiple rounds of cell division4,5. To stop hemorrhaging, however, this model is limited, as the major vessels must be ligated leaving an abnormal environment for regeneration. Many...

The ability to regenerate an organ, or to restore form and function, has been thought to be mostly lost over evolutionary time. The regenerative potential of the adult mammalian liver after acute chemical or physical injury has been found to involve the mobilization of all remaining hepatocytes resulting in waves of hypertrophy and few rounds of cell division, resulting in a functional but architecturally different organ1,2,3,4,5. Recently, studies have begun to characterize the regenerative response of neonatal mammalian organs to injury within the first week of life6,7,8. These studies have shown that when injured during neonatal development, certain mammalian organs respond with morphological regeneration instead of compensatory growth or fibrosis7,8.
Recent studies have shown that regeneration of both global structure and function occurs during the early neonatal period6,7,8. Established liver injury protocols involve chemical injury or administration of ethanol9,10,11, acetaminophen12,13,14,15, carbon tetrachloride16,17,18,19, 70% partial hepatectomy4,20,21, or removal of the left and median lobes. Chemical administration leads to hepatocyte cell death, but often leaves micro- and macro-structures intact. Morphologic regeneration cannot be readily studied in this context, as the overall hepatic architecture was not obliterated. The 70% partial hepatectomy involves suture ligation of the major vessels, which is necessary to stop bleeding, but leaves a non-physiologic environment with permanent disruption of vasculature. Furthermore, this method has only been used on adult rodents, and its application to neonates is technically extremely difficult. With this in mind, we developed a method in which 20-30% of the apex of the left lobe is removed in a newborn P0 mouse (Figure 1A-1B). This method is surgically conservative, minimally invasive, not technically challenging, and leads to gross loss of morphology without the ligation of vasculature, leaving room for regeneration to occur. The resulting step-by-step protocol, described below, allows for any researcher to perform a partial lobular hepatectomy on neonatal mice in order to study mammalian neonatal regeneration in the early stages of post-natal life. This method also has clear applications to comparative studies in regenerative medicine and stem cell biology, as it can be used in the liver during later stages of life.
The most common acute liver injury studies are chemically-induced damage, adult liver amputation, or 70% partial hepatectomy. Chemical damage often involves intravenous, intraperitoneal or oral administration of acetaminophen, carbon tetrachloride, or ethanol, and is a relatively easy and non-invasive injury model. As previously discussed, chemical damage results in hepatocyte cell death, but often leaves stroma and parenchyma structures intact, making it difficult to make claims about morphologic regeneration. Chemical damage often centers on hepatic vessels, making it a useful technique to study site and cell-specific injury, but also makes it difficult to interrogate, at the whole organ level, other populations that may be situated further from vessels and that may contribute to regeneration. Despite these limitations, chemical damage still remains a useful and highly physiologically relevant injury model.
Adult 70% partial hepatectomy involves the removal of the left and median lobes following ligation of hepatic vasculature. The response to hepatectomy has been well characterized: the amputated liver 14 days post 70% partial hepatectomy develops a grossly different architecture from that of the original undamaged lobe, as the hepatocytes of the remaining right and caudate lobes undergo hypertrophy and a few rounds of cell division4,5. This makes up lost mass and function, but fails to regenerate the two amputated lobes, and therefore does not replace gross morphology. As a result, the injury response to 70% partial hepatectomy is useful to study compensatory growth mechanisms with limited regeneration.
Here, we fully describe a protocol for a neonatal partial lobular hepatectomy. The procedure involves appropriate animal selection and preparation, surgical field preparation, surgery, and recovery. Optimization and adaptation of each of these steps may be required for different applications of the protocol.
We have extensively performed and optimized this protocol on wild type C57BL/6J pups (JAX 000664), however, to study different cell populations and mechanisms of regeneration, we also used various transgenic animals including mice harboring various Cre and CreERT2 transgenes and/or knock-ins (Axin2CreERT2 JAX 018867, and Sox9CreERT2 JAX 018829) in combination with fluorescent reporters, such as the Rainbow and mTmG systems (R26VT2/GK3, R26mT/mG)22,23. We found no need to change this methodology for different mouse strains, as no differences in survival outcomes or regenerative potential were observed.
In addition to using different animal strains, we also performed partial lobular hepatectomies on neonatal mice treated with small molecules, such as 4-hydroxy-tamoxifen and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Dimethyl sulfoxide (DMSO) and ethanol were used as solvents, as it was found that corn oil was a significant cause of morbidity. We otherwise found that intraperitoneal administration of small molecules did not affect survival or regenerative outcomes. We predict that this protocol will be adapted for use with other small molecules to interrogate various aspects of regeneration.
Neonatal mouse surgeries can be technically challenging and may require special expertise in animal handling and microscopic dissection. Animal husbandry expertise is necessary to avoid maternal cannibalism following surgery and during the immediate recovery period.

<table>
	<tbody>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mother with litter of day 0 neonatal pups (any strain)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surrogate mother and surrogate litter (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Standard Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Serum (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Providine-iodine or equivalent antiseptic solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td>ZEMSDV4L MFR # 435421-9901-000</td>
			<td></td>
		</tr>
		<tr>
			<td>3mm straight spring micro scissors</td>
			<td>Vannas</td>
			<td>72932-01</td>
			<td></td>
		</tr>
		<tr>
			<td>5SF Forceps</td>
			<td>Dumont</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Kelly forceps</td>
			<td>Grainger</td>
			<td>17-050G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sunbeam</td>
			<td>000771-810-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Labs</td>
			<td>0044-5260-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>1205S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze, 10.16 x 10.16cm</td>
			<td>Fisher Scientific</td>
			<td>13-761-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Standard Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 monocryl sutures</td>
			<td>Ethicon</td>
			<td>MCP489G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>&#160;S35839</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid, Plain, 110V</td>
			<td>Drummond</td>
			<td>4-000-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Mettler Toledo NewClassic ME Analytical Balances</td>
			<td>Fisher Scientific</td>
			<td>01-912-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Cost Induction Chamber</td>
			<td>Kent Scientific</td>
			<td>SOMNO-0730</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial lobular hepatectomy: a surgical model for morphologic liver regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Figure 1A details a general timeline of the neonatal partial lobular hepatectomy (schematic in Figure 1B), and the expected length of time to wait until regeneration is observed. Subtle regeneration of the left lobe can be observed 7-14 days post surgery. Full regeneration was often observed after 56 days post surgery. Mice should show no signs of physiologic abnormalities after surgery.
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Watch this Scientific Journal Video about Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration at JoVE.com

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

The overall goal of this surgical procedure is to induce morphologic liver regeneration via partial lobular hepatectomy. This method can answer key questions in the fields of stem cell biology and regenerative medicine about morphologic liver regeneration and liver specific stem cells. The main advantages of this technique are that it is minimally invasive and does not require chemical insults or cauterization.Prior to touching the animals, rub the bedding from the mother's cage onto the gloves. Then transfer all of the pups and some bedding from the mother's cage into an empty cage on top of a heating pad. Next, place a pup under a dissecting microscope.After confirming the appropriate level of sedation by lack of response to toe pinch, use a small gauze pad to disinfect the posterior abdominal wall of the animal with betadine. Use micro dissecting scissors and forceps to make a left midclavicular half centimeter incision immediately below the ribcage. Use the forceps to gently separate the skin.Then make a second deeper incision into the peritoneal cavity and use the back blunt edge of the micro dissecting scissors and forceps to apply gentle lateral pressure to both sides of the abdomen, forcing the apex of the left lobe out of the peritoneal cavity. When the left apex of the left lobe is visible, amputate the appropriate experimental amount of tissue from the lobe and place the tissue into a 1.5 milliliter tube of PBS on ice. Using a rolled piece of gauze, gently return the left lobe to the peritoneal cavity.Remove the gauze when the bleeding stops and wet the surgical site and surrounding area with gauze soaked with PBS. Close the surgical site with sutures and a running stitch. Then gently but thoroughly roll a PBS soaked gauze over the pup and wound site ensuring no blood or betadine remains.Allow the pup to recover near gauze pads exposed to the mother's feces and urine. When all of the pups have been hepatectomized return all of the animals and bedding to the mother's cage. After weighing the lobes, fix tissue samples in 2%paraformaldehyde for one hour at room temperature.At the end of the fixation, transfer the tissues into individual freezing molds containing optimal cutting temperature medium for freezing over dry ice. And acquire 7-10 micrometer sections of each lobe using any standard cryostat. Check the pups daily for at least 56 days to confirm that their wounds remain closed and inject analgesia 24 and 48 hours post surgery.At the appropriate experimental end point harvest and weigh the liver from each pup on block. Then carefully separate each individual lobe at its proximal attachment and compare the mass of the amputated left lobe to the mass of the whole liver to determine the extent of regeneration in each lobe. Subtle regeneration of the left lobe can be observed 7-14 days post surgery and full regeneration is often observed after 56 days.With no significant difference in hepatocyte areas in the regenerated liver revealed by histological analysis. Further, neonatal mouse injections with EdU indicates a significant increase in the number of proliferating cells in the injured regenerating left lobe compared to uninjured controls suggesting that cell proliferation contributes to left lobe regeneration in neonates. Hepatectomy in post natal day 14 juvenile mice results in a decreased regeneration in the left lobe with an increased compensation from the uninjured lobes, however.Indicating that by 14 days the injury response to acute resection switches from lobe specific regeneration to global compensation. Once mastered, this technique can be completed within 15 minutes if performed properly. While attempting this procedure, it is important to be as careful as possible so not to cause extra stress to the mother or the pups.Following this procedure, other methods such as serial hepatecomies or chronic liver injuries can be done to further assess the regenerative potential of the liver. After its development, this technique paved the way for researchers in the field of stem cell biology to explore liver regeneration in the neonatal mouse. After watching this video, you should have a good understanding of how to complete a lobe specific controlled amputation of the neonatal mouse liver.Don't forget that working with isoflurane is extremely hazardous and precautions such as a face mask and gloves should always be taken when performing this procedure.

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11.9K visualizações.  Stanford University School of Medicine. The overall goal of this surgical procedure is to induce morphologic liver regeneration via partial lobular hepatectomy. This method can answer key questions in the fields of stem cell biology and regenerative medicine about morphologic liver regeneration and liver specific stem cells. The main advantages of this technique are that it is minimally invasive and does not require chemical insults or cauterization.Prior to touching the animals, rub the bedding from the mother's cage onto t...