Extended 78% Hepatectomy in a Mouse Surgical Model

Summary

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.

Abstract

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.

Introduction

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 grafts^1,2,3,4. 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 injury^1,3.

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 syndrome^6,7.

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 ± 1.9%), median lobe (ML; 26.2 ± 1.9%), right upper (also called right superior) lobe (RUL; 16.6 ± 1.4%), right lower (also called right inferior) lobe (RLL; 14.7 ± 1.4%), and caudate lobe (CL, 8.1 ± 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 lethality^1,8,9.

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 ± 5.2%) of the total liver mass. Our choice of removing the LL and ML separately, and not "en bloc" 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 cava^{1,10,11,12,13,14}. 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 failure^15,16. 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.

Protocol

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

1. Shave the mouse abdomen from the mid-sternum to the suprapubic region with clippers.
2. 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.
3. Administer 1.2 mg/kg buprenorphine extended-release (ER) subcutaneously for postoperative analgesia. Place the mouse supine with the forelimbs and hindlegs extended and secure the limbs with tape; then, prepare a sterile field for surgery.
   NOTE: Ensure the forelimbs are relaxed when secured so respiration is not hindered.
4. Prepare the abdomen with warm sterile saline and betadine swabs, alternating between each swab 3 times. Drape the abdomen in a sterile fashion.

2. Hepatectomy

1. Make a vertical midline laparotomy incision through the skin from the xiphoid process to the suprapubic region using 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.
2. 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.
3. 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's capsule, is very fragile and easy to bruise and lacerate.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. Discontinue anesthesia and monitor the mouse until it regains consciousness and can ambulate normally.

3. Postoperative care

1. 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.
2. 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.

Results

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 ...

Discussion

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 lethali...

Materials

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