Name: extended 78% hepatectomy in a mouse surgical model
Uploaded: 2024-05-24T13:20:00.000+00:00
Duration: 5 min 25 s
Description: Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of ...

This work was supported by NIH R01 grants DK063275 and HL086741 to CF. PB and TA are recipients of an NRSA fellowship from the NHLBI T32 training grant HL007734.

The methods described within this procedure protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) at the Beth Israel Deaconess Medical Center (BIDMC). All experiments were completed in accordance and compliance with IACUC and the BIDMC animal research facility guidelines.
1. Mouse preoperative preparation
<ol>
	<li>Shave the mouse abdomen from the mid-sternum to the suprapubic region with clippers.</li>
	<li>Induce general anesthesia with 1-4% isoflurane in 100% oxygen. When anesthetized, place the mouse supine on the surgical field with a heating pad underneath. Before making an incision, firmly pinch the toe to ensure the pedal reflex is absent (if present, the animal will respond). Adjust the anesthetic level as needed to achieve a state of general anesthesia. 
	NOTE: Titrate isoflurane as needed to maintain adequate general anesthesia.</li>
	<li>Administer 1.2 mg/kg buprenorphine extended-release (ER) subcutaneously for postoperative analgesia. Place the mouse supine with the forelimbs&#160;and hindlegs extended and secure the limbs with tape; then, prepare a sterile field for surgery. 
	NOTE: Ensure the forelimbs&#160;are relaxed when secured so respiration is not hindered.</li>
	<li>Prepare the abdomen with warm sterile saline and betadine swabs, alternating between each swab 3 times. Drape the abdomen in a sterile fashion.</li>
</ol>
2. Hepatectomy
<ol>
	<li>Make a vertical midline laparotomy incision through the skin from the xiphoid process to the suprapubic region using&#160;a scalpel. Next, incise through the linea alba with sharp scissors to enter the peritoneal cavity and extend this incision to the length of the skin incision. 
	NOTE: It is safer to first incise the linea alba at the subxiphoid region where the liver is deep to the abdominal wall to avoid injury to the underlying bowel.</li>
	<li>Retract the abdominal wall laterally using appropriate retractors; then, clamp the xiphoid process with a hemostat and retract the sternum superiorly to expose the liver.</li>
	<li>Retract the liver inferiorly to expose the falciform ligament and then, transect the ligament along the length of the liver using sharp scissors. Retract the liver superiorly towards the thorax to expose the hepatogastric ligament and intrahepatic lobe ligaments and transect these structures using sharp micro scissors. 
	NOTE: Retraction should be performed very gently with moist cotton-tipped applicators as the liver, encapsulated by the Glisson&#39;s capsule, is very fragile and easy to bruise and lacerate.</li>
	<li>Retract the median lobe superiorly while keeping the left lobe in its original anatomic position. Wrap a 5-0 silk suture around the superior-medial part of the LL. Reflect the LL superiorly towards the thorax to expose the undersurface of the lobe, bring together the suture ends at the base of the lobe, and tie the suture at the base. Ensure that the suture does not obstruct the blood flow in the inferior vena cava (IVC) or the portal vein before tying the suture to ligate the LL. 
	NOTE: It is best to tie this suture while the LL is reflected superiorly towards the thorax so that the portal triad is well exposed during ligation. This facilitates resection of the lobe close to the base without compromising adjacent structures.</li>
	<li>Resect the LL just distal to the suture tie using sharp scissors and ensure that a small cuff of tissue (~2 mm) separates the suture from the edge of the resected lobe. Confirm hemostasis.</li>
	<li>Reflect the median lobe superiorly towards the thorax, place a 5-0 silk suture around the base of the ML, and return the ML to its original anatomic position. Approximate the suture ends over the base of the superior aspect of the ML and ensure tie them at the base of the lobe. Resect the ligated ML, leaving a small cuff of remnant tissue around the suture tie. Confirm hemostasis.</li>
	<li>Mobilize the liver from right to left to expose the right upper and lower lobes and carefully reflect these lobes medially and inferiorly. Wrap a 5-0 suture over the superior-medial aspect of the RUL to ensure the suture encircles the RUL base and then, reflect the RUL towards the thorax. Wrap the suture under the RUL and tie the ends close to its base, then resect it, leaving a small cuff of remnant tissue around the suture tie. 
	NOTE: Tying too proximal at the base of the RUL can compromise the blood supply to the RLL, which can result in ischemia of the RLL and death of the mouse within 24 h postoperatively. In contrast, tying too distally from the RUL base decreases the amount of resected of liver mass, thereby increasing postoperative survival rates beyond what is expected.</li>
	<li>Return the remaining liver to its resting anatomic position and ensure hemostasis. If required, apply pressure with gauze to areas of minor bleeding at resected liver margins.</li>
	<li>Close the midline abdominal wall (fascia and muscle layers) using a 5-0 polyglactin suture in an uninterrupted fashion. Close the skin incision with staples or 5-0 monofilament sutures.</li>
	<li>Discontinue anesthesia and monitor the mouse until it regains consciousness and can ambulate normally.</li>
</ol>
3. Postoperative care
<ol>
	<li>Observe the mouse postoperatively to ensure appropriate recovery (i.e. the mouse is awake, alert, and ambulatory within the cage) and pain control. Examine the mouse every 2 h up until 6 h after the procedure, and then daily. 
	NOTE: It is expected that the mouse will move slower within the cage postoperatively. The mouse should recover in an isolated cage from other mice and only returned to the company of other mice when it is fully recovered.</li>
	<li>Administer warmed normal saline injections (0.1-1.0 mL, subcutaneous or intraperitoneal) if the mouse becomes hypovolemic from insensible fluid or excessive blood loss from surgery.</li>
</ol>

Mouse Extended Hepatectomy Model for Liver Regeneration Studies

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There are no conflicts of interest to disclose.

To successfully perform an extended 78% hepatectomy causing 50% lethality in mice, it is critical that each liver lobe is precisely resected. This level of competency and precision can only be achieved if the procedure is performed repeatedly. The training curve varies between operators but typically requires 3-6 months of practice. A liver resection that removes less than 78% of the TLM would result in higher survival rates, while a liver resection that removes greater than 78% of the TLM would result in greater lethality. Each lobe resection is challenging, albeit not to the same extent.
The left lobe is the easiest to reliably resect. The LL base is narrow, and when reflected superiorly, the operator can easily identify the liver base and tie the suture in the same position with near-identical lobe resection volume each time. The median lobe has the widest base when compared to the LL and RUL. Therefore, it requires that the operator carefully estimate where the suture needs to be placed and carefully approximate the suture ends at the base of the ML prior to resecting the lobe. When the ML is tied too proximally, the suture may compromise venous liver drainage or impede blood return from the IVC to the heart. When the ML is tied too distally, insufficient liver mass is resected, and the risk of hemorrhage at the resection margin increases since the ML base is wider. The right upper lobe is perhaps the most difficult to reliably resect. The anatomic position of the RUL posteriorly in the peritoneal cavity makes it difficult to wrap the suture completely around its base, which could result in an incomplete resection of this lobe. In contrast, if the suture is tied too proximally at the RUL base, blood supply to the right lower lobe might be jeopardized, causing ischemia of this RLL and increasing the likelihood of postoperative mortality.
Other critical elements to minimize procedure-related risks include the minimization of general anesthesia (e.g., Isoflurane) to reduce toxicity and ensuring adequate hemostasis after each lobe resection to limit postoperative hemorrhage. It is important to consider that the extended 78% hepatectomy is preferably performed in adult 8-12-week-old mice, as older mice can exhibit more variability in survival rates due to their greater body mass and reduced regenerative liver capacity, while younger mice might suffer greater technical complications due to the smaller size of their liver and a higher rate of anesthesia-related complications. We surmise that the observed 50% lethality following 78% hepatectomy corresponds to intrinsic single mouse characteristics which relate to subtle anatomical variations in the relative percentage of each liver lobe mass relative to total liver mass between individual animals. The 78% hepatectomy in mice represents an anatomic threshold at which only 50% of the animals can timely and successfully regenerate and survive while the other 50% fail to do so and die. We also acknowledge that subtle differences in liver manipulation might be associated with different degrees of liver damage, and hence skew this fine balance towards failure to regenerate and death22.
Albeit, the most limiting factor remains the mastery of the surgical procedure itself, which can only come with practice. Practice is essential to ensure a reproducible outcome through precise mapping of the sutures at the base of each liver lobe. One cautionary note is that individual variations in the liver anatomy amongst mice - which are rare - may require some modification in technique. Other interventions that should be considered on a case-per-case basis to improve success include administration of normal saline boluses in case of high insensible fluid losses or significant hemorrhage, and prolonged manual pressure or electrocautery at the liver margin in instances of persisting hemorrhage.
In summary, the extended 78% hepatectomy in a mouse model is a valuable technique for translational science research. Extensive training is critical to achieve a technically successful outcome associated with this procedure. Mice are not only a preferred small animal species that are easy-to-handle and relatively inexpensive, but also available in a number of well-studied inbred strains in addition to an ever-growing number of genetically modified lines (transgenic, knockout (KO), cell-type specific, and conditional KO), enabling fine mechanistic studies3,9,23. In addition to genetically modified mice, various pathologies, including non-alcoholic fatty liver disease, cirrhosis, and diabetes that are known to influence survival and outcomes after liver resection, can be easily induced in mice24,25,26,27,28,29,30,31.
As mentioned earlier, the classic 2/3 PH remains extremely valuable but does not recapitulate the high lethality that is associated with extended liver resection for cancer or small-for-size syndrome following liver transplantation when the liver mass is anatomically or functionally inadequate (such as in fatty livers)7,32,33,34. When properly performed, this extended 78% hepatectomy results in 50% postoperative death, which better reflects clinical reality such as following extensive liver resections for trauma or cancer and in the context of small-for-size syndrome following transplantation of marginal liver grafts, and also after mere liver surgery in patients with severe non-alcoholic steatohepatitis (NASH) or cirrhosis16,32. This mouse model represents a highly valuable and necessary proof-of-concept step to test novel therapeutic strategies to improve outcomes in all these conditions. Any positive results in mice are bound to substantially decrease the number of animals required to conduct pre-translational large animal studies prior to clinical translation of innovative therapies.

Mouse and rat surgical liver resection models, first described in 1931, are the most common experimental models utilized to study the molecular basis of liver regeneration. They could also be useful in translational science research to test and develop strategies to improve outcomes following extended liver resection or transplantation of suboptimal liver grafts1,2,3,4. Partial hepatectomies (PH) in mice entail the removal of approximately 2/3 (66%) of the total liver mass (TLM), which when performed in healthy animals have exceptional outcomes5. The procedure is short in duration, easily reproducible due to little variation in mouse liver anatomy, and postoperative survival typically nears 100%1.
Partial 2/3 hepatectomy encompassing the resection of the left lobe (LL) and median lobe (ML) allows for the residual lobes to regenerate relatively unimpeded by lobar inflammation or restriction of hepatic inflow and outflow. Rather, increased portal venous flow and subsequently shear stress on liver sinusoidal endothelial cells following PH result in sustained upregulation of endothelial nitric oxide synthase (eNOS) expression and subsequent nitric oxide (NO) release, which contribute to the priming of hepatocytes for proliferation and liver regeneration3. Outcomes commonly studied after 2/3 PH in disease models such as non-alcoholic fatty liver disease or in specific genetic backgrounds include risk of acute liver failure, qualitative and quantitative measures of the liver regenerative capacity, and other biologic responses to stress or traumatic injury1,3.
However, a mouse model mimicking functional or anatomical small-for-size syndrome, as it occurs following extended liver resection for cancer or transplantation of marginal (steatosis or prolonged ischemic time) or partial (split or from living donor liver) liver grafts, remains to be well-established. To address this need, models of more extensive liver resections that extend beyond the maintenance of a minimal (and functional) liver mass are required to model small-for-size liver syndrome and the heightened mortality that is associated with this syndrome6,7.
Mouse liver anatomy exhibits minimal variation. The mouse liver is comprised of five lobes, each accounting for the following percentage of the total liver mass: left lobe (LL; 34.4 &#177; 1.9%), median lobe (ML; 26.2 &#177; 1.9%), right upper (also called right superior) lobe (RUL; 16.6 &#177; 1.4%), right lower (also called right inferior) lobe (RLL; 14.7 &#177; 1.4%), and caudate lobe (CL, 8.1 &#177; 1.0%)1,5. Each lobe is supplied by a portal triad, including a branch of the hepatic artery, a branch of the portal vein, and a bile duct5. Historically, several techniques were described to perform a 2/3 PH by resecting the LL and the ML. These include 1) the classical technique that consists of a single ligature en bloc at the base of each of the resected lobes; 2) the hemostatic clip technique, using titanium clips applied at the base of the resected lobes; 3) a vessel-oriented parenchyma-preserving technique, using piercing sutures proximal to the clamp; and 4) a vessel-oriented microsurgical technique, whereby the portal vein and hepatic artery branches are ligated prior to lobe resection1. While each technique has relative strengths and weaknesses, none yields higher lethality1,8,9.
In this study, we present a novel method for extended 78% PH in mice. In this model, three of five liver lobes, including the LL, ML, and RUL, are removed separately using a ligature technique (Figure 1). This procedure results in the resection of approximately 78% (77.2 &#177; 5.2%) of the total liver mass. Our choice of removing the LL and ML separately, and not &#34;en bloc&#34; as in the classical PH technique, minimizes complications that are associated with en bloc resection of these two lobes, such as suprahepatic vena cava stenosis and heightened risk of necrosis of the remaining lobes when the single ligature is applied too close to the vena cava1,10,11,12,13,14. This is crucial before moving to the final step of this procedure to remove the RUL. This extensive hepatectomy in 8-12 weeks old, wild-type C57BL/6 mice causes 50% lethality within 1 week of surgery due to failed liver regeneration causing fulminant liver failure15,16. This mouse model of heightened lethality following extended 78% hepatectomy appropriately recapitulates the pathophysiology of small-for-size syndrome and enables the development and testing of novel strategies to improve outcomes.

<table><tbody><tr><td>2 x 2 Gauze</td><td>Covidien</td><td>2146</td><td>Surgery: dissection</td></tr><tr><td>5-O Nylon Monofilament Suture</td><td>Oasis</td><td>50-118-0631</td><td>Surgery: Skin closure</td></tr><tr><td>5-O Silk Suture</td><td>Fine Science Tools</td><td>18020-50</td><td>Surgery: liver lobe ligation</td></tr><tr><td>5-O Vicryl Suture</td><td>Ethicon</td><td>NC9335902</td><td>Surgery: Abdominal wall closure</td></tr><tr><td>Addson Forceps</td><td>Braintree Scientific</td><td>FC028</td><td>Surgery: dissection</td></tr><tr><td>Alcohol Swabs (2)</td><td>BD</td><td>326895</td><td>Disinfectant</td></tr><tr><td>Buprenorphine Extended Release Formulation&amp;nbsp;</td><td>Zoopharm</td><td>N/A</td><td>Analgesia</td></tr><tr><td>Cordless Trimmer</td><td>Braintree Scientific</td><td>CLP-9868-14</td><td>Shaving</td></tr><tr><td>Curved Forceps</td><td>Braintree Scientific</td><td>FC0038</td><td>Surgery: dissection</td></tr><tr><td>Hemostat</td><td>Braintree Scientific</td><td>FC79-1</td><td>Surgery: dissection</td></tr><tr><td>Isoflurane Inhalant Anesthetic&amp;nbsp;</td><td>Patterson Veterinary</td><td>RXISO-250</td><td>General Anesthesia</td></tr><tr><td>Magnet Fixator (2-slot) (2)</td><td>Braintree Scientific</td><td>ACD-001</td><td>Surgery: to hold small retractors</td></tr><tr><td>Magnet Fixator (4-slot)&amp;nbsp;</td><td>Braintree Scientific</td><td>ACD-002</td><td>Surgery: to hold small retractors</td></tr><tr><td>Microscissors</td><td>Braintree Scientific</td><td>SC-MI 151</td><td>Surgery: dissection</td></tr><tr><td>Operating tray</td><td>Braintree Scientific</td><td>ACD-0014</td><td>Surgery: for establishment of surgical field&amp;nbsp;</td></tr><tr><td>Povidone Iodine 10% Swabstick (2)</td><td>Medline</td><td>MDS093901ZZ</td><td>Disinfectant</td></tr><tr><td>Scalpel (15-blade)</td><td>Aspen Surgical Products</td><td>371615</td><td>Surgery: dissection</td></tr><tr><td>Sharp Scissors (Curved)</td><td>Braintree Scientific</td><td>SC-T-406</td><td>Surgery: dissection</td></tr><tr><td>Sharp Scissors (Straight)</td><td>Braintree Scientific</td><td>SC-T-405</td><td>Surgery: dissection</td></tr><tr><td>Small Cotton-Tipped Applicators</td><td>Fisher Scientific</td><td>23-400-118</td><td>Surgery: dissection</td></tr><tr><td>Tissue Forceps (Straight x2)</td><td>Braintree Scientific</td><td>FC1001</td><td>Surgery: dissection</td></tr><tr><td>Warming Pad (18&quot; x 26&quot;)</td><td>Stryker</td><td>TP 700</td><td>Warming</td></tr><tr><td>Warming Pad Pump</td><td>Stryker</td><td>TP 700</td><td>Warming</td></tr><tr><td>Wire Handle Retractor (2)&amp;nbsp;</td><td>Braintree Scientific</td><td>ACD-005</td><td>Surgery: to facilitate exposure of peritoneal cavity</td></tr><tr><td>Xenotec Isoflurane Small Animal Anesthesia System</td><td>Braintree Scientific</td><td>EZ-108SA</td><td>General Anesthesia: Contains Isoflurane vaborizer &amp;amp; console, Induction chamber, Regulator/Hose, Facemask (M)</td></tr></tbody></table>

extended 78&#37; hepatectomy in a mouse surgical model

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of disease models. In the classical partial 2/3 hepatectomy in mice, two of the five liver lobes, namely the left and median lobes representing approximately 66% of the liver mass, are resected en bloc with an expected postoperative survival of 100%. More aggressive partial hepatectomies are technically more challenging and hence, have seldom been used in mice. Our group has developed a mouse model of an extended hepatectomy technique in which three of the five liver lobes, including the left, median, and right upper lobes, are resected separately to remove approximately 78% of the total liver mass. This extended resection, in otherwise healthy mice, leaves a remnant liver that cannot always sustain adequate and timely regeneration. Failure to regenerate ultimately results in 50% postoperative lethality within 1 week due to fulminant hepatic failure. This procedure of extended 78% hepatectomy in mice represents a unique surgical model for the study of small-for-size syndrome and the evaluation of therapeutic strategies to improve liver regeneration and outcomes in the setting of liver transplantation or extended liver resection for cancer.

A successful extended 78% hepatectomy is expected to induce 50% mortality within 1 week in healthy adult mice aged 8-12 weeks16. When properly performed, minimal blood loss is expected. Residual bleeding that persists can be controlled by manual pressure. Perioperative death within 24 h of surgery is often caused by technical errors. Technical failures include inadvertent injury to large blood vessels causing intractable intraoperative hemorrhage; significant postoperative hemorrhage often due to residual bleeding from the resected liver margins; injury to surrounding structure such as inadvertent ligation of the adjacent portal triad, the portal vein, or the IVC; ischemia of the RLL secondary to ligation of the RUL too proximally to the lobe base; and complications from general anesthesia. Signs of hepatic failure include progressive lethargy, hair clumping, anorexia, and hypoglycemia, which often become apparent within 24 h of surgery.
The two expected outcomes after extended 78% hepatectomy are either survival or death. In the first scenario, the mouse recovers appropriately after surgery, resumes normal activity within 72 h, and survives beyond 7 days. Laparotomy performed 9-10 days postoperatively demonstrates full recovery of presurgery liver mass4,16,17,18. The second outcome would be mortality within 2-7 days postoperatively. The mouse may show initial signs of recovery in the first 12 h after surgery but worsens relatively quickly afterwards due to the development of fulminant liver failure16. The mouse exhibits signs of stress, weight loss, and progressive lethargy until death. Examples of unexpected outcomes include death from technical complications or mouse survival rates well above or below 50% after extended 78% hepatectomy.
Our laboratory pioneered this extended 78% hepatectomy and validated its usefulness in an earlier manuscript showing that extended 78% hepatectomy in 8-week-old healthy BALB/c mice results in 50% lethality, which could be abrogated if the mouse liver was preengineered to express A20 (Tumor Necrosis Factor Inducible Protein 3 [TNFAIP3]), delivered intravenously using a recombinant adenovirus16. In this study by Longo et al., a healthy BALB/c mouse underwent a 78% percent hepatectomy 5 days following the administration of a recombinant adenovirus vector expressing human A20 (rAd.A20) or the control &#946;-Galactosidase (rAd.&#946;Gal). An additional non-treated control group was also included. Following 78% hepatectomy, Longo et al. observed that 6 of the 12 (50%) non-treated mice survived the procedure (Figure 2)16.
Two determinants of proficiency include 1) limiting technical complications, as listed above, chief of which being RLL ischemia, causing early death, and 2) ensuring sufficient mobilization of the RUL, without which one fails to appreciate the full size of the RUL and hence, fails to properly resect it. This reduces the amount of liver mass resected and therefore, improves the overall survival rate well above the expected 50%. In the initial period of training of the investigator/author, 10 of 15 (67%) healthy adult male and female CD1 and C57BL/6 mice aged 11-21 weeks survived 1 week following 78% hepatectomy (3 technical deaths excluded) (Table 1)19,20,21. Further training in the context of an appreciative understanding of mouse liver anatomy to improve RUL mobilization and facilitate an adequate resection, measured by estimated percent hepatectomy, was essential to achieve the expected 50% survival rate following 78% hepatectomy. After technical proficiency was felt to be fully achieved, 8 of 16 mice (50%; 1 technical death excluded) survived 1 week following 78% hepatectomy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66528/66528fig01.jpg" /> 
Figure 1: Mouse hepatectomy positioning and liver anatomy. (Left) Visual representation of mouse positioning for laparotomy and extended 78% hepatectomy. The mouse is depicted supine with a midline laparotomy incision. (Right) Mouse liver anatomy depicted from below with colored lines delineating resection sites. The left lobe, median lobe, and right upper lobes are resected in a sequential fashion following suture ligation at their base. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66528/66528fig01large.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/66528/66528fig02.jpg" /> 
Figure 2: Survival advantage following extended (78%) hepatectomy of mice engineered to overexpress A20. Survival data following 78% extended hepatectomy in control untreated (non-infected NI), rAd.&#946;Gal, and rAd.A20-treated mice. Overexpression of A20 in mouse livers yielded a significant survival advantage compared with NI (50%) and rAd.&#946;Gal controls (13%) (n = 12 mice/group). This figure is from Longo et al.16. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66528/66528fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Survived</td>
			<td>Deceased</td>
			<td>Technical Failure</td>
			<td>Total</td>
			<td>Estimated % Hepatectomy</td>
			<td>Survival Rate</td>
		</tr>
		<tr>
			<td>Pre-Proficiency</td>
			<td>10</td>
			<td>5</td>
			<td>3</td>
			<td>18</td>
			<td>68 &#177; 3.9%</td>
			<td>10/15 (66%)</td>
		</tr>
		<tr>
			<td>Post-Proficiency</td>
			<td>8</td>
			<td>8</td>
			<td>1</td>
			<td>17</td>
			<td>79 &#177; 2.4%</td>
			<td>8/16 (50%)</td>
		</tr>
	</tbody>
</table>
Table 1: Extended 78% hepatectomy training results. Survival results within 1 week following extended 78% hepatectomy in healthy adult CD1 and C57BL/6 mice aged 11-21 weeks. Technical proficiency was self-determined via observable improvements in technical success and adequate resection of the right upper lobe, as calculated by estimated percent hepatectomy (Mean &#177; SEM).

Author Spotlight: Evaluating Therapeutic Strategies to Enhance Liver Regeneration

The mouse model of partial 2/3 (66%) hepatectomy is well described in the literature, but more extended hepatectomies mimicking small-for-size syndrome after liver transplantation have seldom been used. We describe an extended 78% hepatectomy procedure in a mouse model that results in approximately 50% postoperative lethality in healthy mice.

Extended 78&#37; Hepatectomy in a Mouse Surgical Model

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of ...

extended-78-hepatectomy-in-a-mouse-surgical-model

Universidad San Sebastian

Research

JoVE Journal

Biology

1.5K Views.  Beth Israel Deaconess Medical Center and Harvard Medical School. The mouse model of partial 2/3 (66%) hepatectomy is well described in the literature, but more extended hepatectomies mimicking small-for-size syndrome after liver transplantation have seldom been used. We describe an extended 78% hepatectomy procedure in a mouse model that results in approximately 50% postoperative lethality in healthy mice. 

Video: Author Spotlight: Evaluating Therapeutic Strategies to Enhance Liver Regeneration

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Cancer Research

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.

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 ...

Developmental Biology

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Medicine

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

Studying Liver Regeneration of ALLPS in Animal Models for Staged Hepatectomy

A Mouse Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Procedure Aided by Microscopy

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative mortality, severely impacting patient outcomes. In a robust hepatic environment, the future liver remnant (FLR) must exceed 25%, and in cases of cirrhosis, this requirement increases to over 40%. The inadequacy of FLR is currently a major obstacle in the progression of hepatic surgery.
Traditional methods to enhance FLR hypertrophy mainly focus on portal vein embolization (PVE), but its effectiveness is considerably limited. In recent years, there have been numerous reports on a novel biphasic hepatectomy method involving hepatic partitioning and portal vein ligation, known as associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). ALPPS surpasses PVE in efficiently and considerably inducing FLR hypertrophy. However, the detailed mechanisms driving ALPPS-facilitated hepatic regeneration are not fully understood. Thus, replicating ALPPS in animal models is crucial to thoroughly investigate the molecular mechanisms of hepatic regeneration, offering valuable theoretical and practical insights.

Author Spotlight: Advancing Liver Regeneration Research through ALPPS Mouse Model

Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) has been shown to result in remarkable liver regeneration after several weeks post first-stage surgery. Animal models that have undergone ALPPS have been developed to explore potential liver regeneration ability and achieve favorable clinical outcomes.

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative ...

Extended 78% Hepatectomy in a Mouse Surgical Model

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