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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	<li>Martins, P. N., Theruvath, T. P., Neuhaus, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+partial+hepatectomies.">Rodent models of partial hepatectomies.</a> Liver Int. 28 (1), 3-11 (2008).</li><li>Higgins, G., Anderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+pathology+of+the+liver+I.+Restoration+of+the+liver+of+the+white+rat+following+partial+surgical+removal.">Experimental pathology of the liver I. Restoration of the liver of the white rat following partial surgical removal.</a> Arch Pathol. 12, 186-202 (1931).</li><li>Koniaris, L. G., McKillop, I. H., Schwartz, S. I., Zimmers, T. A. <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> J Am Coll Surg. 197 (4), 634-659 (2003).</li><li>Fausto, N., Campbell, J. S., Riehle, K. J. <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> Hepatology. 43 (2), S45-S53 (2006).</li><li>Inderbitzin, D., 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=Magnetic+resonance+imaging+provides+accurate+and+precise+volume+determination+of+the+regenerating+mouse+liver.">Magnetic resonance imaging provides accurate and precise volume determination of the regenerating mouse liver.</a> J Gastrointest Surg. 8 (7), 806-811 (2004).</li><li>Clavien, P. A., 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=What+is+critical+for+liver+surgery+and+partial+liver+transplantation:+size+or+quality.">What is critical for liver surgery and partial liver transplantation: size or quality.</a> Hepatology. 52 (2), 715-729 (2010).</li><li>Dahm, F., Georgiev, P., Clavien, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-for-size+syndrome+after+partial+liver+transplantation:+definition,+mechanisms+of+disease+and+clinical+implications.">Small-for-size syndrome after partial liver transplantation: definition, mechanisms of disease and clinical implications.</a> Am J Transplant. 5 (11), 2605-2610 (2005).</li><li>Hori, T., 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=Simple+and+reproducible+hepatectomy+in+the+mouse+using+the+clip+technique.">Simple and reproducible hepatectomy in the mouse using the clip technique.</a> World J Gastroenterol. 18 (22), 2767-2774 (2012).</li><li>Kamali, C., 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=Extended+liver+resection+in+mice:+state+of+the+art+and+pitfalls-a+systematic+review.">Extended liver resection in mice: state of the art and pitfalls-a systematic review.</a> Eur J Med Res. 26 (1), 6 (2021).</li><li>Mitchell, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+and+well-tolerated+method+for+2/3+partial+hepatectomy+in+mice.">A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.</a> Nat Protoc. 3 (7), 1167-1170 (2008).</li><li>Borowiak, 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=Met+provides+essential+signals+for+liver+regeneration.">Met provides essential signals for liver regeneration.</a> Proc Natl Acad Sci U S A. 101 (29), 10608-10613 (2004).</li><li>Boyce, S., Harrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+methodology+of+partial+hepatectomy+in+the+mouse.">A detailed methodology of partial hepatectomy in the mouse.</a> Lab Anim (NY). 37 (11), 529-532 (2008).</li><li>Greene, A. K., Puder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+hepatectomy+in+the+mouse:+technique+and+perioperative+management.">Partial hepatectomy in the mouse: technique and perioperative management.</a> J Invest Surg. 16 (2), 99-102 (2003).</li><li>Mitchell, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erratum:+A+reproducible+and+well-tolerated+method+for+2/3+partial+hepatectomy+in+mice.">Erratum: A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.</a> Nat Protoc. 9 (6), 1532 (2014).</li><li>Studer, P., 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=Significant+lethality+following+liver+resection+in+A20+heterozygous+knockout+mice+uncovers+a+key+role+for+A20+in+liver+regeneration.">Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration.</a> Cell Death Differ. 22 (12), 2068-2077 (2015).</li><li>Longo, C. R., 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=A20+protects+mice+from+lethal+radical+hepatectomy+by+promoting+hepatocyte+proliferation+via+a+p21waf1-dependent+mechanism.">A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism.</a> Hepatology. 42 (1), 156-164 (2005).</li><li>Michalopoulos, G. K., DeFrances, M. 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+regeneration.">Liver regeneration.</a> Science. 276 (5309), 60-66 (1997).</li><li>Diehl, A. M., Rai, R. M. <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.+3.+Regulation+of+signal+transduction+during+liver+regeneration.">Liver regeneration. 3. Regulation of signal transduction during liver regeneration.</a> FASEB J. 10 (2), 215-227 (1996).</li><li>. A comparison of selected organ weights and clinical pathology parameters in male and female CD-1 and CByB6F1 hybrid mice 12-14 weeks in age Available from: <a target="_blank" href="https://www.criver.com/sites/default/files/resources/doc_a/AComparisonofSelectedOrganWeightsandClinicalPathologyParametersinMaleandFemaleCD-1andCByB6F1HybridMice12-14WeeksinAge.pdf">https://www.criver.com/sites/default/files/resources/doc_a/AComparisonofSelectedOrganWeightsandClinicalPathologyParametersinMaleandFemaleCD-1andCByB6F1HybridMice12-14WeeksinAge.pdf</a> (2023)</li><li>CD-1&#174; IGS mouse. Charles River Laboratories Available from: <a target="_blank" href="https://www.criver.com/products-services/find-model/cd-1r-igs-mouse?region=3611">https://www.criver.com/products-services/find-model/cd-1r-igs-mouse?region=3611</a> (2023)</li><li>C57BL/6J mouse organ weight. The Jackson Laboratory Available from: <a target="_blank" href="https://www.jax.org/de/-/media/jaxweb/files/jax-mice-and-services/b6j-data-summary.xlsx">https://www.jax.org/de/-/media/jaxweb/files/jax-mice-and-services/b6j-data-summary.xlsx</a> (2023)</li><li>Inderbitzin, D., 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=Regenerative+capacity+of+individual+liver+lobes+in+the+microsurgical+mouse+model.">Regenerative capacity of individual liver lobes in the microsurgical mouse model.</a> Microsurgery. 26 (6), 465-469 (2006).</li><li>Zhou, X., 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=L-carnitine+promotes+liver+regeneration+after+hepatectomy+by+enhancing+lipid+metabolism.">L-carnitine promotes liver regeneration after hepatectomy by enhancing lipid metabolism.</a> J Transl Med. 21 (1), 487 (2023).</li><li>Linecker, 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=Omega-3+fatty+acids+protect+fatty+and+lean+mouse+livers+after+major+hepatectomy.">Omega-3 fatty acids protect fatty and lean mouse livers after major hepatectomy.</a> Ann Surg. 266 (2), 324-332 (2017).</li><li>Haber, B. 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A., 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=Impact+of+steatosis+on+perioperative+outcome+following+hepatic+resection.">Impact of steatosis on perioperative outcome following hepatic resection.</a> J Gastrointest Surg. 7 (8), 1034-1044 (2003).</li><li>Ma, K., 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=A+mesenchymal-epithelial+transition+factor-agonistic+antibody+accelerates+cirrhotic+liver+regeneration+and+improves+mouse+survival+following+partial+hepatectomy.">A mesenchymal-epithelial transition factor-agonistic antibody accelerates cirrhotic liver regeneration and improves mouse survival following partial hepatectomy.</a> Liver Transpl. 28 (5), 782-793 (2022).</li><li>Hori, T., 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=Simple+and+sure+methodology+for+massive+hepatectomy+in+the+mouse.">Simple and sure methodology for massive hepatectomy in the mouse.</a> Ann Gastroenterol. 24 (4), 307-318 (2011).</li><li>Ramsey, H. 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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 lethali...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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)&#160;</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&#160;</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&#34; x 26&#34;)</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)&#160;</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 &#38; 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 ...

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 ...

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Research

JoVE Journal

Biology

1.3K 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

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

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.

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 ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...